Methods and systems for sorting droplets and beads

ABSTRACT

Methods and systems for sorting droplets are provided. In some cases, occupied droplets may be sorted from unoccupied droplets. In some cases, singularly occupied droplets may be sorted from unoccupied droplets and multiply occupied droplets. Methods and systems for sorting cell beads are provided. In some cases, cell beads may be sorted from particles unoccupied with cell derivatives. In some cases, singularly occupied cell beads may be sorted from unoccupied particles and multiply occupied cell beads. Methods and systems for selectively polymerizing droplets based on occupancy and size of the droplets are provided.

CROSS-REFERENCE

This application is a continuation of International Application No.PCT/US2018/033280, filed May 17, 2018, which claims the benefit of U.S.Provisional Patent Application No. 62/508,219, filed May 18, 2017, eachof which is entirely incorporated herein by reference.

BACKGROUND

A sample may be processed for various purposes, such as identificationof a type of moiety within the sample. The sample may be a biologicalsample. Biological samples may be processed, such as for detection of adisease (e.g., cancer) or identification of a particular species. Thereare various approaches for processing samples, such as polymerase chainreaction (PCR) and sequencing.

Biological samples may be processed using various reaction environments,such as partitions. Partitions may be wells or droplets. Droplets orwells may enable biological samples to be partitioned and processedseparately. For example, such droplets may be fluidically isolated fromother droplets, enabling accurate control of respective environments inthe droplets.

A plurality of droplets can be generated such that one or more dropletsinclude cells and/or particles. The cells and/or particles can be ofinterest for use in various (e.g., single cell) applications, such asnucleic acid amplification and/or sequencing applications.

SUMMARY

As recognized herein, when a plurality of droplets is generated, somedroplets may not include any particles, such as cells and beads. Aparticle may be a bead, such as a gel bead and/or a cell bead. Aparticle may be a biological particle, such as a cell or cellderivative. A particle, such as a gel bead, may have a molecular barcodecoupled thereto. Thus, recognized herein is a need to sort the pluralityof droplets into a first subset of droplets that include particles and asecond subset of droplets that do not. In some instances, when aplurality of cell beads is generated, some particles generated with theplurality of cell beads may not include any cells (e.g., non-cell bead).Recognized herein is a need to isolate the plurality of cell beads, suchas by sorting a plurality of particles into a first subset of particlesthat include cells (e.g., cell beads) and a second subset of particlesthat do not.

In some aspects, the systems and methods for sorting described hereinmay yield an output comprising mostly singularly occupied droplets(containing a single particle of interest). For example, at least about90%, 95%, 96%, 97%, 98%, 99%, or more of a plurality of droplets may besingularly occupied droplets. Droplets may be sorted, such as by (i)introducing field-attractable particles (e.g., magnetic particles) intothe droplets and subjecting the droplets to a field (e.g., magneticfield), (ii) subjecting the droplets to a pressure pulse and separatingthe droplets based on hydrodynamic forces, and/or (iii) directing thedroplets to interface physical structures (e.g., having apertures) in aflow path of the droplets and separating the droplets based onmechanical properties (e.g., deformability) of the droplets.

In some aspects, the systems and methods for sorting described hereinmay yield an output comprising mostly singularly occupied cell beads(containing a single cell). For example, at least about 90%, 95%, 96%,97%, 98%, 99%, or more of a population of beads (or particles) may besingularly occupied cell beads. Cell beads may be isolated (or sorted),such as by (i) generating cell beads with field-attractable particles(e.g., magnetic particles), such as by polymerizing the dropletscontaining the field-attractable particles, and subjecting the cellbeads to a field (e.g., magnetic field), (ii) subjecting the cell beadsto a pressure pulse and separating the cell beads via hydrodynamicforces, and/or (iii) directing the cell beads to interface physicalstructures (e.g., having apertures) in a flow path of the cell beads andseparating the cell beads based on mechanical properties (e.g.,deformability) of the cell beads. In some cases, already sorteddroplets, which are mostly singularly occupied droplets (e.g.,containing a single cell), may be polymerized to generate cell beadsthat are mostly singularly occupied. In some cases, a plurality ofdroplets may be selectively polymerized, such that mostly (or only)singularly occupied droplets are polymerized to generate cell beads thatare mostly singularly occupied.

Provided herein are methods and systems for sorting droplets that canisolate droplets that include biological particles (e.g., a cell) and/orother particles (e.g., gel beads, cell beads, etc.) from droplets thatdo not include biological particles and/or other particles. The methodsand systems may isolate droplets that are singularly occupied fromdroplets that are non-singularly occupied, such as from unoccupieddroplets or multiply occupied droplets. In another aspect, providedherein are methods and systems that can isolate particles that includecells (e.g., cell beads) from particles that do not include cells. Themethods and systems may isolate cell beads that are singularly occupiedfrom particles that are non-singularly occupied, such as from unoccupiedparticles or multiply occupied cell beads. The isolated droplets (thatinclude biological particles and/or other particles) and/or isolatedcell beads (that include cells) can be subject to further applications,such as nucleic acid amplification and/or sequencing. Beneficially, suchpre-sorting may increase efficiency of downstream applications bysignificantly saving time and resources (e.g., valuable reagents).

The methods and systems generally operate by generating a plurality ofdroplets such that each of the plurality of droplets comprisesfield-attractable particles. A given droplet in the plurality ofdroplets may or may not include one or more particles (e.g., biologicalparticles, beads, etc.). Thus, the plurality of droplets comprisingfield attractable particles can comprise a first subset of droplets thatinclude one or more particles and a second subset of droplets that donot include any particles. A given droplet in the first subset ofdroplets that include one or more particles can comprise a sufficientlydiscrepant number or concentration of field-attractable particles than agiven droplet in the second subset of droplets that do not include anyparticles such that when the plurality of droplets is subject to anelectric or magnetic field, the first subset of droplets and the secondsubset of droplets are separated from each other. In some cases, whenthe plurality of droplets is subjected to an electric or magnetic field,singularly occupied droplets may be separated from unoccupied dropletsand otherwise multiply occupied droplets.

In some instances, a plurality of particles may be generated withfield-attractable particles, such as by polymerizing the plurality ofdroplets comprising the field-attractable particles. A given particlemay or may not include a cell. Thus, the plurality of particlescomprising field attractable particles may comprise a first subset ofparticles (e.g., cell beads) that include cells and a second subset ofparticles that does not include cells. A given cell bead in the firstsubset of particles can comprise a sufficiently discrepant number orconcentration of field-attractable particles than a given particle inthe second subset of particles, such that when the plurality ofparticles is subject to an electric or magnetic field, the first subsetof particles and the second subset of particles are separated from eachother. In some cases, when the plurality of particles is subjected to anelectric or magnetic field, singularly occupied cell beads may beseparated from unoccupied particles and otherwise multiply occupied cellbeads.

In some instances, a plurality of droplets can be generated withoutfield-attractable particles. A given droplet in the plurality ofdroplets may or may not include one or more particles. Thus, theplurality of droplets can comprise a first subset of droplets thatinclude one or more particles and a second subset of droplets that donot include any particles. The plurality of droplets can be subject to apressure pulse and the first subset of droplets and the second subset ofdroplets can be separated from each other via hydrodynamic forces. Insome cases, the plurality of droplets can be subject to an electricfield, and the first subset and the second subset of droplets can beseparated via dielectrophoresis. In some cases, singularly occupieddroplets may be separated from unoccupied droplets and otherwisemultiply occupied droplets.

In some instances, a plurality of particles can be generated withoutfield-attractable particles. A given particle in the plurality particlesmay or may not include one or more cells. Thus, the plurality ofparticles can comprise a first subset of particles (e.g., cell beads)that include one or more cells and a second subset of particles that donot include any cells. The plurality of particles can be subject to apressure pulse and the first subset of particles and the second subsetof particles can be separated from each other via hydrodynamic forces.In some cases, the plurality of particles can be subject to an electricfield, and the first subset and the second subset can be separated viadielectrophoresis. In some cases, singularly occupied cell beads may beseparated from unoccupied particles and otherwise multiply occupied cellbeads.

In some instances, a plurality of droplets comprising a first subset ofdroplets that include one or more particles and a second subset ofdroplets that do not include any particles can be sorted via a passivemechanism based on mechanical properties of the droplets, such as therespective deformability properties of the droplets. When the pluralityof droplets is directed to pass through one or more apertures, eachaperture having a size smaller than a minimum dimension of a droplet,only deforming droplets may pass through the apertures and non-deformingdroplets may be trapped on the apertures. Unoccupied droplets may havehigher deformability and/or lower surface tension properties compared tooccupied droplets, thus allowing occupied droplets to be trapped on oneor more apertures, and allowing unoccupied droplets to pass through theone or more apertures, thereby separating the first subset and secondsubset of droplets from the plurality of droplets.

In some instances, a plurality of particles comprising a first subset ofparticles (e.g., cell beads) that include one or more cells and a secondsubset of particles that do not include any particles can be sorted viaa passive mechanism based on mechanical properties of the particles,such as the respective deformability properties (or rigidity) of theparticles. When the plurality of particles is directed to pass throughone or more apertures, each aperture having a size smaller than aminimum dimension of a particle, only deforming particles may passthrough the apertures and non-deforming particles may be trapped on theapertures. Unoccupied particles (e.g., not having cells or theirderivatives) may have higher deformability and/or lower surface tensionproperties compared to cell beads, thus allowing cell beads to betrapped on one or more apertures, and allowing unoccupied particles topass through the one or more apertures, thereby separating the firstsubset and second subset of particles from the plurality of particles.

In an aspect, provided is a method for sorting droplets, comprising: (a)bringing a first phase in contact with a second phase to generate aplurality of droplets, wherein the first phase and second phase areimmiscible, wherein the plurality of droplets comprisesfield-attractable particles and wherein (i) a first subset of theplurality of droplets includes biological particles or particles havingcoupled thereto molecular barcodes, and (ii) a second subset of theplurality of droplets does not include the biological particles; (b)directing the plurality of droplets along a first channel towards anintersection of the first channel with a second channel and a thirdchannel; and (c) subjecting the plurality of droplets comprising thefield-attractable particles to an electric or magnetic field underconditions sufficient to separate at least a portion of the first subsetof the plurality of droplets from at least a portion of the secondsubset of the plurality of droplets, wherein upon separation, the atleast the portion of the first subset of the plurality of droplets flowsalong the second channel and the at least the portion of the secondsubset of the plurality of droplets flows along the third channel.

In some embodiments, the second subset of the plurality of droplets doesnot include the particles having coupled thereto molecular barcodes.

In some embodiments, a concentration of the field-attractable particlesin the second subset of the plurality of droplets is substantiallyuniform.

In some embodiments, each droplet of the first subset of the pluralityof droplets comprises less field attractable particles than each dropletof the second subset of the plurality of droplets. In some embodiments,wherein the electric or magnetic field induces forces on the secondsubset of the plurality of droplets that is greater than forces inducedon the first subset of the plurality of droplets.

In some embodiments, the field-attractable particles are magnetic-fieldattractable particles. In some embodiments, the field-attractableparticles are paramagnetic particles.

In some embodiments, the field-attractable particles are electric-fieldattractable particles. In some embodiments, the field-attractableparticles are conductive particles.

In some embodiments, the first subset of the plurality of dropletsincludes biological particles and the particles having coupled theretomolecular barcodes. In some embodiments, the particles having coupledthereto molecular barcodes are beads. In some embodiments, the beads aregel beads.

In some embodiments, the method further comprises, subsequent to (c),subjecting nucleic acid molecules derived from the biological particlesin the first subset to nucleic acid sequencing. In some embodiments, themethod further comprises, subsequent to (c), subjecting the first subsetof the plurality of droplets to nucleic acid amplification conditions toyield amplification products of the nucleic acid molecules from thebiological particles in the first subset. In some embodiments, themethod further comprises subjecting the amplification products tonucleic acid sequencing.

In some embodiments, the conditions of the electric or magnetic fieldsufficient to separate the at least the portion of the first subset ofthe plurality of droplets and the at least the portion of the secondsubset of the plurality of droplets are determined based at least inpart on a ratio between sizes of the plurality of droplets and sizes ofthe biological particles and/or particles having coupled theretomolecular barcodes in the first subset of the plurality of droplets.

In some embodiments, the plurality of droplets is directed along thefirst channel using a pressure pulse.

In some embodiments, the molecular barcodes are releasably coupled tothe particles.

In some embodiments, the method further comprises subjecting individualdroplets of the first subset of the plurality of droplets to a stimulusto facilitate polymerization in the biological particles. In someembodiments, the stimulus is an optical stimulus. In some embodiments,the optical stimulus a laser or ultraviolet light. In some embodiments,the stimulus is a chemical stimulus. In some embodiments, the stimulusis applied prior to the intersection. In some embodiments, the stimulusis applied along the first channel. In some embodiments, the stimulus isapplied along the second channel. In some embodiments, the methodfurther comprises (i) detecting the individual droplets and (ii)subjecting the individual droplets to the stimulus upon detecting theindividual droplets.

In some embodiments, the biological particles are cells enclosed withinor comprising a gel or polymer matrix.

In some embodiments, the first subset comprises a third subset ofdroplets each comprising a single biological particle and a fourthsubset of droplets each comprising multiple biological particles, themethod further comprising: directing the first subset of the pluralityof droplets along the second channel towards a second intersection ofthe second channel with a fourth channel and a fifth channel, andsubjecting the first subset to an electric or magnetic field underconditions sufficient to separate at least a portion of the third subsetfrom at least a portion of the fourth subset, wherein upon separation,the at least the portion of the third subset of droplets flows along afourth channel and the at least the portion of the fourth subset ofdroplets flows along a fifth channel.

In another aspect, provided is a system for sorting droplets,comprising: a fluid flow path comprising a first channel, a secondchannel and a third channel; a fluid flow unit that is configured tosubject a plurality of droplets to flow along the first channel, whereinthe plurality of droplets is generated upon bringing a first phase incontact with a second phase, wherein the first phase and second phaseare immiscible, wherein the plurality of droplets comprisesfield-attractable particles, and wherein (i) a first subset of theplurality of droplets includes biological particles or particles havingcoupled thereto molecular barcodes, and (ii) a second subset of theplurality of droplets does not include the biological particles; a fieldapplication unit that is configured to apply an electric or magneticfield; and a controller operatively coupled to the fluid flow unit andthe field application unit, wherein the controller is programmed to (i)direct the fluid flow unit to subject the plurality of droplets to flowalong the first channel to an intersection of the first channel with thesecond channel and the third channel, and (ii) direct the fieldapplication unit to subject the plurality of droplets comprising thefield-attractable particles to the electric or magnetic field underconditions sufficient to separate at least a portion of the first subsetof the plurality of droplets from at least a portion of the secondsubset of the plurality of droplets, wherein upon separation, the atleast the portion of the first subset of the plurality of droplets flowsalong the second channel and the at least the portion of the secondsubset of the plurality of droplets flows along the third channel.

In some embodiments, the second subset of the plurality of droplets doesnot include the particles having coupled thereto molecular barcodes.

In some embodiments, the field application unit is configured to applythe electric field. In some embodiments, the field application unit isconfigured to apply the magnetic field. In some embodiments, the fieldapplication unit is configured to apply the electric field and magneticfield.

In some embodiments, the field-attractable particles are magnetic-fieldattractable particles. In some embodiments, the field-attractableparticles are paramagnetic particles.

In some embodiments, the field-attractable particles are electric-fieldattractable particles. In some embodiments, the field-attractableparticles are conductive particles.

In some embodiments, the fluid flow unit includes at least one pump thatis configured to provide negative pressure. In some embodiments, thefluid flow unit includes at least one compressor that is configured toprovide positive pressure.

In some embodiments, the fluid flow unit is configured to apply apressure pulse to direct the plurality of droplets along the firstchannel.

In some embodiments, the fluid flow unit is configured to apply apressure pulse to direct the first or second subset of the plurality ofdroplets along the second channel or third channel, respectively.

In some embodiments, the controller is programmed to direct the fluidflow unit to subject the first subset of the plurality of droplets to apressure pulse at the intersection to subject the first subset of theplurality of droplets to flow along the second channel.

In some embodiments, the fluid flow unit includes an actuator that isconfigured to subject the plurality of droplets to flow.

In some embodiments, the controller is programmed to determine theconditions of the electric or magnetic field sufficient to separate theat least the portion of the first subset of the plurality of dropletsand the at least the portion of the second subset of the plurality ofdroplets based at least in part on a ratio between sizes of theplurality of droplets and/or sizes of the biological particles orparticles having coupled thereto molecular barcodes in the first subsetof the plurality of droplets.

In some embodiments, each droplet of the first subset of the pluralityof droplets comprises less field attractable particles than each dropletof the second subset of the plurality of droplets. In some embodiments,wherein the electric or magnetic field induces forces on the secondsubset of the plurality of droplets that is greater than forces inducedon the first subset of the plurality of droplets.

In some embodiments, the biological particles are cells enclosed withinor comprising a gel or polymer matrix.

In another aspect, provided is a non-transitory computer-readable mediumcomprising machine-executable code that, upon execution by one or morecomputer processors, implements a method for sorting droplets,comprising: (a) bringing a first phase in contact with a second phase togenerate a plurality of droplets, wherein the first phase and secondphase are immiscible, wherein the plurality of droplets comprisesfield-attractable particles, and wherein (i) a first subset of theplurality of droplets includes biological particles or particles havingcoupled thereto molecular barcodes, and (ii) a second subset of theplurality of droplets does not include the biological particles; (b)directing the plurality of droplets along a first channel towards anintersection of the first channel with a second channel and a thirdchannel; and (c) subjecting the plurality of droplets comprising thefield-attractable particles to an electric or magnetic field underconditions sufficient to separate at least a portion of the first subsetof the plurality of droplets from at least a portion of the secondsubset of the plurality of droplets, wherein upon separation, the atleast the portion of the first subset of the plurality of droplets flowsalong the second channel and the at least the portion of the secondsubset of the plurality of droplets flows along the third channel.

In another aspect, provided is a method for sorting droplets,comprising: (a) bringing a first phase in contact with a second phase togenerate a plurality of droplets, wherein the first phase and secondphase are immiscible, and wherein (i) a first subset of the plurality ofdroplets includes biological particles or particles, which particlescomprise molecular barcodes coupled thereto, and (ii) a second subset ofthe plurality of droplets does not include the biological particles; (b)directing the plurality of droplets along a first channel towards anintersection of the first channel with a second channel and a thirdchannel; and (c) at the intersection, subjecting the plurality ofdroplets to a pressure pulse under conditions sufficient to separate atleast a portion of the first subset of the plurality of droplets from atleast a portion of the second subset of the plurality of droplets,wherein upon separation, the at least the portion of the first subset ofthe plurality of droplets flows along the second channel and the atleast the portion of the second subset of the plurality of dropletsflows along the third channel.

In some embodiments, the second subset of the plurality of droplets doesnot include the particles having coupled thereto molecular barcodes.

In some embodiments, the pressure pulse induces forces on the secondsubset of the plurality of droplets that is greater than forces inducedon the first subset of the plurality of droplets.

In some embodiments, the first subset of the plurality of dropletsincludes biological particles and the particles having coupled theretomolecular barcodes. In some embodiments, the particles having coupledthereto molecular barcodes are beads. In some embodiments, the beads aregel beads.

In some embodiments, the method further comprises, subsequent to (c),subjecting nucleic acid molecules derived from the biological particlesin the first subset to nucleic acid sequencing. In some embodiments, themethod further comprises, subsequent to (c), subjecting the first subsetof the plurality of droplets to nucleic acid amplification conditions toyield amplification products of the nucleic acid molecules from thebiological particles in the first subset. In some embodiments, themethod further comprises subjecting the amplification products tonucleic acid sequencing.

In some embodiments, the molecular barcodes are releasably coupled tothe particles.

In some embodiments, the method further comprises subjecting individualdroplets of the first subset of the plurality of droplets to a stimulusto facilitate polymerization in the biological particles. In someembodiments, the method further comprises (i) detecting the individualdroplets and (ii) subjecting the individual droplets to the stimulusupon detecting the individual droplets.

In some embodiments, the biological particles are cells enclosed withinor comprising a gel or polymer matrix.

In another aspect, provided is a system for sorting droplets,comprising: a fluid flow path comprising a first channel, a secondchannel and a third channel; a fluid flow unit that is configured tosubject a plurality of droplets to flow along the first channel, whereinthe plurality of droplets is generated upon bringing a first phase incontact with a second phase, wherein the first phase and second phaseare immiscible, and wherein (i) a first subset of the plurality ofdroplets includes biological particles or particles having coupledthereto molecular barcodes, and (ii) a second subset of the plurality ofdroplets does not include the biological particles; a pressureapplication unit that is configured to apply a pressure pulse; and acontroller operatively coupled to the fluid flow unit and the pressureapplication unit, wherein the controller is programmed to (i) direct thefluid flow unit to subject the plurality of droplets to flow along thefirst channel to an intersection of the first channel with the secondchannel and the third channel, and (ii) direct the pressure applicationunit to subject the plurality of droplets to the pressure pulse underconditions sufficient to separate at least a portion of the first subsetof the plurality of droplets from at least a portion of the secondsubset of the plurality of droplets, wherein upon separation, the atleast the portion of the first subset of the plurality of droplets flowsalong the second channel and the at least the portion of the secondsubset of the plurality of droplets flows along the third channel.

In another aspect, provided is a non-transitory computer-readable mediumcomprising machine-executable code that, upon execution by one or morecomputer processors, implements a method for sorting droplets,comprising: (a) bringing a first phase in contact with a second phase togenerate a plurality of droplets, wherein the first phase and secondphase are immiscible, and wherein (i) a first subset of the plurality ofdroplets includes biological particles or particles having coupledthereto molecular barcodes, and (ii) a second subset of the plurality ofdroplets does not include the biological particles; (b) directing theplurality of droplets along a first channel towards an intersection ofthe first channel with a second channel and a third channel; and (c)subjecting the plurality of droplets to a pressure pulse underconditions sufficient to separate at least a portion of the first subsetof the plurality of droplets from at least a portion of the secondsubset of the plurality of droplets, wherein upon separation, the atleast the portion of the first subset of the plurality of droplets flowsalong the second channel and the at least the portion of the secondsubset of the plurality of droplets flows along the third channel.

In another aspect, provided is a method for droplet processing,comprising: (a) bringing a first phase in contact with a second phase togenerate a plurality of droplets, wherein the first phase and secondphase are immiscible, wherein (i) a first subset of the plurality ofdroplets includes biological particles, and (ii) a second subset of theplurality of droplets does not include the biological particles; (b)directing the plurality of droplets along a first channel towards anintersection of the first channel with a second channel and a thirdchannel; (c) prior to the intersection, selectively subjectingindividual droplets of the first subset of the plurality of droplets toa stimulus to facilitate polymerization in the biological particles; and(d) separating at least a portion of the first subset of the pluralityof droplets from at least a portion of the second subset of theplurality of droplets at the intersection, wherein upon separation, theat least the portion of the first subset of the plurality of dropletsflows along the second channel and the at least the portion of thesecond subset of the plurality of droplets flows along the thirdchannel.

In some embodiments, the first subset of the plurality of dropletsinclude particles having coupled thereto molecular barcodes.

In some embodiments, the second subset of the plurality of droplets doesnot include the particles having coupled thereto molecular barcodes.

In some embodiments, the method further comprises (i) detecting theindividual droplets and (ii) selectively subjecting the individualdroplets to the stimulus upon detecting the individual droplets.

In another aspect, provided is a method for sorting droplets,comprising: (a) bringing a first phase in contact with a second phase togenerate a plurality of droplets, wherein the first phase and secondphase are immiscible, wherein the plurality of droplets comprisesfield-attractable particles and wherein the plurality of dropletscomprises (i) a first subset of droplets each including, and not morethan, one biological particle, and (ii) a second subset of droplets eacheither not including any biological particle or including more than onebiological particle; (b) directing the plurality of droplets along afirst channel towards an intersection of the first channel with a secondchannel and a third channel; and (c) subjecting the plurality ofdroplets comprising the field-attractable particles to an electric ormagnetic field under conditions sufficient to separate at least aportion of the first subset of the plurality of droplets from at least aportion of the second subset of the plurality of droplets, wherein uponseparation, the at least the portion of the first subset of theplurality of droplets flows along the second channel and the at leastthe portion of the second subset of the plurality of droplets flowsalong the third channel.

In some embodiments, the first subset of the plurality of dropletsinclude particles having coupled thereto molecular barcodes. In someembodiments, the particles having coupled thereto molecular barcodes arebeads. In some embodiments, the beads are gel beads.

In some embodiments, a concentration of the field-attractable particlesin droplets of the second subset which do not include any biologicalparticle is substantially uniform.

In some embodiments, each droplet of the first subset of the pluralityof droplets comprises (i) less field attractable particles than eachdroplet of the second subset of the plurality of droplets which do notinclude any biological particle, and (ii) more field attractableparticles than each droplet of the second subset of the plurality ofdroplets which includes more than one biological particle. In someembodiments, forces induced by the electric or magnetic field ondroplets of the second subset which do not include any biologicalparticle is greater than forces induced on the first subset, whichforces induced on the first subset are greater than forces induced ondroplets of the second subset which includes more than one biologicalparticle.

In some embodiments, the field-attractable particles are magnetic-fieldattractable particles. In some embodiments, the field-attractableparticles are paramagnetic particles.

In some embodiments, the field-attractable particles are electric-fieldattractable particles. In some embodiments, the field-attractableparticles are conductive particles.

In some embodiments, the method further comprises, subsequent to (c),subjecting nucleic acid molecules derived from the biological particlesin the first subset to nucleic acid sequencing.

In some embodiments, the method further comprises, subsequent to (c),subjecting the first subset of the plurality of droplets to nucleic acidamplification conditions to yield amplification products of the nucleicacid molecules from the biological particles in the first subset. Insome embodiments, the method further comprises subjecting theamplification products to nucleic acid sequencing.

In some embodiments, the conditions of the electric or magnetic fieldsufficient to separate the at least the portion of the first subset ofthe plurality of droplets and the at least the portion of the secondsubset of the plurality of droplets are determined based at least inpart on a ratio between sizes of the plurality of droplets and sizes ofthe biological particles in the first subset of the plurality ofdroplets.

In some embodiments, the plurality of droplets is directed along thefirst channel using a pressure pulse.

In some embodiments, the method further comprises subjecting individualdroplets of the first subset of the plurality of droplets to a stimulusto facilitate polymerization in the biological particles. In someembodiments, the stimulus is applied prior to the intersection. In someembodiments, the method further comprises (i) detecting the individualdroplets and (ii) subjecting the individual droplets to the stimulusupon detecting the individual droplets.

In some embodiments, the biological particles are cells enclosed withinor comprising a gel or polymer matrix.

In another aspect, provided is a method for sorting droplets,comprising: (a) bringing a first phase in contact with a second phase togenerate a plurality of droplets, wherein the first phase and secondphase are immiscible, wherein the plurality of droplets comprises (i) afirst subset of droplets each including, and not more than, onebiological particle, and (ii) a second subset of droplets each eithernot including any biological particle or including more than onebiological particle; (b) directing the plurality of droplets along afirst channel towards an intersection of the first channel with a secondchannel and a third channel; and (c) subjecting the plurality ofdroplets to a pressure pulse under conditions sufficient to separate atleast a portion of the first subset of the plurality of droplets from atleast a portion of the second subset of the plurality of droplets,wherein upon separation, the at least the portion of the first subset ofthe plurality of droplets flows along the second channel and the atleast the portion of the second subset of the plurality of dropletsflows along the third channel.

In some embodiments, the first subset of the plurality of dropletsinclude particles having coupled thereto molecular barcodes. In someembodiments, the particles having coupled thereto molecular barcodes arebeads. In some embodiments, the beads are gel beads.

In some embodiments, forces induced by the pressure pulse on droplets ofthe second subset which do not include any biological particle isgreater than forces induced on the first subset, which forces induced onthe first subset are greater than forces induced on droplets of thesecond subset which includes more than one biological particle.

In some embodiments, the method further comprises, subsequent to (c),subjecting nucleic acid molecules derived from the biological particlesin the first subset to nucleic acid sequencing. In some embodiments, themethod further comprises, subsequent to (c), subjecting the first subsetof the plurality of droplets to nucleic acid amplification conditions toyield amplification products of the nucleic acid molecules from thebiological particles in the first subset. In some embodiments, themethod further comprises subjecting the amplification products tonucleic acid sequencing.

In some embodiments, the method further comprises subjecting individualdroplets of the first subset of the plurality of droplets to a stimulusto facilitate polymerization in the biological particles. In someembodiments, the method further comprises (i) detecting the individualdroplets and (ii) subjecting the individual droplets to the stimulusupon detecting the individual droplets.

In some embodiments, the biological particles are cells enclosed withinor comprising a gel or polymer matrix.

In another aspect, provided is a method for sorting particles,comprising: (a) providing a plurality of particles, wherein theplurality of particles comprises (i) a first subset of particles eachincluding a biological particle from or contents of a plurality of cellsand (ii) a second subset of particles each not including a biologicalparticle from or contents of the plurality of cells; and (b) sorting theplurality of particles, thereby isolating at least a portion of thefirst subset of particles from at least a portion of the second subsetof particles.

In some embodiments, the first subset of particles comprises a thirdsubset of particles each including, but not more than, one biologicalparticle from the plurality of cells and a fourth subset of particleseach including more than one biological particle from the plurality ofcells. In some embodiments, the method further comprises sorting thefirst subset of particles, thereby isolating at least a portion of thethird subset of particles from at least a portion of the fifth subset ofparticles.

In some embodiments, (b) comprises subjecting the plurality of particlesto a magnetic or electric field. In some embodiments, each particle ofthe plurality of particles comprises field-attractable particles.

In some embodiments, wherein (b) comprises subjecting the plurality ofparticles to a pressure pulse.

In another aspect, provided is a method for sorting particles,comprising: (a) providing a plurality of particles generated from aplurality of cells, wherein the plurality of particles comprises (i) afirst subset of particles each including, but not more than, onebiological particle from or contents of a single cell from the pluralityof cells and (ii) a second subset of particles each either not includinga biological particle from or contents of the plurality of cells orincluding more than one biological particle from or contents of theplurality of cells; and (b) sorting the plurality of particles, therebyisolating at least a portion of the first subset of particles from atleast a portion of the second subset of particles.

In some embodiments, (b) comprises subjecting the plurality of particlesto a magnetic or electric field. In some embodiments, each particle ofthe plurality of particles comprises field-attractable particles.

In some embodiments, (b) comprises subjecting the plurality of particlesto a pressure pulse.

In another aspect, provided is a method for processing droplets. Themethod can comprise: providing a plurality of gel beads in a firstphase, wherein the plurality of gel beads comprise (i) molecularbarcodes and (ii) field-attractable particles; and subjecting theplurality of gel beads comprising the field-attractable particles to anelectric or magnetic field under conditions sufficient to separate theplurality of gel beads from at least 50% of the first phase, therebyproviding the plurality of gel beads in a second phase that isimmiscible with respect to the first phase.

In some embodiments, the plurality of gel beads can be separated from atleast 60% of the first phase. In some embodiments, the plurality of gelbeads can be separated from at least 80% of the first phase. In someembodiments, the plurality of gel beads can be separated from at least90% of the first phase.

In some embodiments, the first phase can be an oil phase. In someembodiments, the second phase can be an aqueous phase.

In another aspect, provided is a method for sorting gel beads,comprising: processing a plurality of droplets to generate a pluralityof gel beads, wherein the plurality of droplets comprisesfield-attractable particles and wherein (i) a first subset of theplurality of gel beads includes biological particles or particles havingcoupled thereto molecular barcodes, and (ii) a second subset of theplurality of gel beads does not include the biological particles,directing the plurality of gel beads along a first channel towards anintersection of the first channel with a second channel and a thirdchannel; and subjecting the plurality of gel beads to an electric ormagnetic field under conditions sufficient to separate the first subsetof the plurality of gel beads from the second subset of the plurality ofgel beads, wherein upon separation, the first subset of the plurality ofgel beads flows along the second channel and the second subset of theplurality of gel beads flows along the third channel.

In some embodiments, the processing comprises polymerizing the pluralityof droplets.

In another aspect, provided is a method for sorting gel beads,comprising: processing a plurality of droplets to generate a pluralityof gel beads, wherein (i) a first subset of the plurality of gel beadsincludes biological particles or particles having coupled theretomolecular barcodes, and (ii) a second subset of the plurality of gelbeads does not include the biological particles, directing the pluralityof gel beads along a first channel towards an intersection of the firstchannel with a second channel and a third channel; and at theintersection, subjecting the plurality of gel beads to a pressure pulseunder conditions sufficient to separate the first subset of theplurality of gel beads from the second subset of the plurality of gelbeads, wherein upon separation, the first subset of the plurality of gelbeads flows along the second channel and the second subset of theplurality of gel beads flows along the third channel.

Additional aspects and advantages of the present disclosure will becomereadily apparent to those skilled in this art from the followingdetailed description, wherein only illustrative embodiments of thepresent disclosure are shown and described. As will be realized, thepresent disclosure is capable of other and different embodiments, andits several details are capable of modifications in various obviousrespects, all without departing from the disclosure. Accordingly, thedrawings and description are to be regarded as illustrative in nature,and not as restrictive.

INCORPORATION BY REFERENCE

All publications, patents, and patent applications mentioned in thisspecification are herein incorporated by reference to the same extent asif each individual publication, patent, or patent application wasspecifically and individually indicated to be incorporated by reference.To the extent publications and patents or patent applicationsincorporated by reference contradict the disclosure contained in thespecification, the specification is intended to supersede and/or takeprecedence over any such contradictory material.

BRIEF DESCRIPTION OF THE DRAWINGS

The novel features of the invention are set forth with particularity inthe appended claims. A better understanding of the features andadvantages of the present invention will be obtained by reference to thefollowing detailed description that sets forth illustrative embodiments,in which the principles of the invention are utilized, and theaccompanying drawings (also “Figure” and “FIG.” herein), of which:

FIG. 1 shows an example of a microfluidic channel structure forpartitioning individual biological particles.

FIG. 2A shows an example of a microfluidic channel structure forseparating occupied droplets from unoccupied droplets.

FIG. 2B shows an example of a multi-stage microfluidic channel structurefor separating singularly occupied droplets.

FIG. 3 shows another example of a microfluidic channel structure forseparating occupied droplets from unoccupied droplets.

FIG. 4 shows an example of a microfluidic channel structure forselective polymerization of partitions based on occupancy.

FIG. 5 shows another example of a microfluidic channel structure forselective polymerization of partitions based on occupancy.

FIG. 6 shows an example of a microfluidic channel structure forselective polymerization of partitions based on droplet size.

FIG. 7 shows a flowchart for a method of sorting occupied droplets andunoccupied droplets.

FIG. 8 shows a flowchart for another method of sorting occupied dropletsand unoccupied droplets.

FIG. 9 shows a flowchart for a method of selectively polymerizingoccupied droplets.

FIG. 10 shows a flowchart for a method of selectively polymerizingappropriately sized droplets.

FIG. 11 shows an example of a microfluidic channel structure forseparating occupied droplets from unoccupied droplets.

FIG. 12 shows an example of a microfluidic channel structure fordelivering barcode carrying beads to droplets.

FIG. 13 shows an example of a microfluidic channel structure forco-partitioning biological particles and reagents.

FIG. 14 shows an example of a microfluidic channel structure for thecontrolled partitioning of beads into discrete droplets.

FIG. 15 shows an example of a microfluidic channel structure forincreased droplet generation throughput.

FIG. 16 shows another example of a microfluidic channel structure forincreased droplet generation throughput.

FIG. 17A shows a cross-section view of another example of a microfluidicchannel structure with a geometric feature for controlled partitioning.FIG. 17B shows a perspective view of the channel structure of FIG. 17A.

FIG. 18 shows an example computer control system that is programmed orotherwise configured to implement methods provided herein.

FIG. 19 illustrates an example of a barcode carrying bead.

DETAILED DESCRIPTION

While various embodiments of the invention have been shown and describedherein, it will be obvious to those skilled in the art that suchembodiments are provided by way of example only. Numerous variations,changes, and substitutions may occur to those skilled in the art withoutdeparting from the invention. It should be understood that variousalternatives to the embodiments of the invention described herein may beemployed.

Where values are described as ranges, it will be understood that suchdisclosure includes the disclosure of all possible sub-ranges withinsuch ranges, as well as specific numerical values that fall within suchranges irrespective of whether a specific numerical value or specificsub-range is expressly stated.

The term “barcode,” as used herein, generally refers to a label, oridentifier, that conveys or is capable of conveying information about ananalyte. A barcode can be part of an analyte. A barcode can beindependent of an analyte. A barcode can be a tag attached to an analyte(e.g., nucleic acid molecule) or a combination of the tag in addition toan endogenous characteristic of the analyte (e.g., size of the analyteor end sequence(s)). A barcode may be unique. Barcodes can have avariety of different formats. For example, barcodes can include:polynucleotide barcodes; random nucleic acid and/or amino acidsequences; and synthetic nucleic acid and/or amino acid sequences. Abarcode can be attached to an analyte in a reversible or irreversiblemanner. A barcode can be added to, for example, a fragment of adeoxyribonucleic acid (DNA) or ribonucleic acid (RNA) sample before,during, and/or after sequencing of the sample. Barcodes can allow foridentification and/or quantification of individual sequencing-reads.

The term “real time,” as used herein, can refer to a response time ofless than about 1 second, a tenth of a second, a hundredth of a second,a millisecond, or less. The response time may be greater than 1 second.In some instances, real time can refer to simultaneous or substantiallysimultaneous processing, detection or identification.

The term “subject,” as used herein, generally refers to an animal, suchas a mammal (e.g., human) or avian (e.g., bird), or other organism, suchas a plant. For example, the subject can be a vertebrate, a mammal, arodent (e.g., a mouse), a primate, a simian or a human. Animals mayinclude, but are not limited to, farm animals, sport animals, and pets.A subject can be a healthy or asymptomatic individual, an individualthat has or is suspected of having a disease (e.g., cancer) or apre-disposition to the disease, and/or an individual that is in need oftherapy or suspected of needing therapy. A subject can be a patient. Asubject can be a microorganism or microbe (e.g., bacteria, fungi,archaea, viruses).

The term “genome,” as used herein, generally refers to genomicinformation from a subject, which may be, for example, at least aportion or an entirety of a subject's hereditary information. A genomecan be encoded either in DNA or in RNA. A genome can comprise codingregions (e.g., that code for proteins) as well as non-coding regions. Agenome can include the sequence of all chromosomes together in anorganism. For example, the human genome ordinarily has a total of 46chromosomes. The sequence of all of these together may constitute ahuman genome.

The terms “adaptor(s)”, “adapter(s)” and “tag(s)” may be usedsynonymously. An adaptor or tag can be coupled to a polynucleotidesequence to be “tagged” by any approach, including ligation,hybridization, or other approaches.

The term “sequencing,” as used herein, generally refers to methods andtechnologies for determining the sequence of nucleotide bases in one ormore polynucleotides. The polynucleotides can be, for example, nucleicacid molecules such as deoxyribonucleic acid (DNA) or ribonucleic acid(RNA), including variants or derivatives thereof (e.g., single strandedDNA). Sequencing can be performed by various systems currentlyavailable, such as, without limitation, a sequencing system byIllumina®, Pacific Biosciences (PacBio®), Oxford Nanopore®, or LifeTechnologies (Ion Torrent®). Alternatively or in addition, sequencingmay be performed using nucleic acid amplification, polymerase chainreaction (PCR) (e.g., digital PCR, quantitative PCR, or real time PCR),or isothermal amplification. Such systems may provide a plurality of rawgenetic data corresponding to the genetic information of a subject(e.g., human), as generated by the systems from a sample provided by thesubject. In some examples, such systems provide sequencing reads (also“reads” herein). A read may include a string of nucleic acid basescorresponding to a sequence of a nucleic acid molecule that has beensequenced. In some situations, systems and methods provided herein maybe used with proteomic information.

The term “bead,” as used herein, generally refers to a particle. Thebead may be a solid or semi-solid particle. The bead may be a gel bead.The gel bead may include a polymer matrix (e.g., matrix formed bypolymerization or cross-linking). The polymer matrix may include one ormore polymers (e.g., polymers having different functional groups orrepeat units). Polymers in the polymer matrix may be randomly arranged,such as in random copolymers, and/or have ordered structures, such as inblock copolymers. Cross-linking can be via covalent, ionic, orinductive, interactions, or physical entanglement. The bead may be amacromolecule. The bead may be formed of nucleic acid molecules boundtogether. The bead may be formed via covalent or non-covalent assemblyof molecules (e.g., macromolecules), such as monomers or polymers. Suchpolymers or monomers may be natural or synthetic. Such polymers ormonomers may be or include, for example, nucleic acid molecules (e.g.,DNA or RNA). The bead may be formed of a polymeric material. The beadmay be magnetic or non-magnetic. The bead may be rigid. The bead may beflexible and/or compressible. The bead may be disruptable ordissolvable. The bead may be a solid particle (e.g., a metal-basedparticle including but not limited to iron oxide, gold or silver)covered with a coating comprising one or more polymers. Such coating maybe disruptable or dissolvable.

The term “sample,” as used herein, generally refers to a biologicalsample of a subject. The biological sample may comprise any number ofmacromolecules, for example, cellular macromolecules. The sample may bea cell sample. The sample may be a cell line or cell culture sample. Thesample can include one or more cells. The sample can include one or moremicrobes. The biological sample may be a nucleic acid sample or proteinsample. The biological sample may also be a carbohydrate sample or alipid sample. The biological sample may be derived from another sample.The sample may be a tissue sample, such as a biopsy, core biopsy, needleaspirate, or fine needle aspirate. The sample may be a fluid sample,such as a blood sample, urine sample, or saliva sample. The sample maybe a skin sample. The sample may be a cheek swab. The sample may be aplasma or serum sample. The sample may be a cell-free or cell freesample. A cell-free sample may include extracellular polynucleotides.Extracellular polynucleotides may be isolated from a bodily sample thatmay be selected from the group consisting of blood, plasma, serum,urine, saliva, mucosal excretions, sputum, stool and tears.

The term “biological particle,” as used herein, generally refers to adiscrete biological system derived from a biological sample. Thebiological particle may be a macromolecule. The biological particle maybe a small molecule. The biological particle may be a virus. Thebiological particle may be a cell or derivative of a cell. Thebiological particle may be an organelle. The biological particle may bea rare cell from a population of cells. The biological particle may beany type of cell, including without limitation prokaryotic cells,eukaryotic cells, bacterial, fungal, plant, mammalian, or other animalcell type, mycoplasmas, normal tissue cells, tumor cells, or any othercell type, whether derived from single cell or multicellular organisms.The biological particle may be a constituent of a cell. The biologicalparticle may be or may include DNA, RNA, organelles, proteins, or anycombination thereof. The biological particle may be or may include amatrix (e.g., a gel or polymer matrix) comprising a cell or one or moreconstituents from a cell (e.g., cell bead), such as DNA, RNA,organelles, proteins, or any combination thereof, from the cell. Thebiological particle may be obtained from a tissue of a subject. Thebiological particle may be a hardened cell. Such hardened cell may ormay not include a cell wall or cell membrane. The biological particlemay include one or more constituents of a cell, but may not includeother constituents of the cell. An example of such constituents is anucleus or an organelle. A cell may be a live cell. The live cell may becapable of being cultured, for example, being cultured when enclosed ina gel or polymer matrix, or cultured when comprising a gel or polymermatrix.

The term “macromolecular constituent,” as used herein, generally refersto a macromolecule contained within or from a biological particle. Themacromolecular constituent may comprise a nucleic acid. In some cases,the biological particle may be a macromolecule. The macromolecularconstituent may comprise DNA. The macromolecular constituent maycomprise RNA. The RNA may be coding or non-coding. The RNA may bemessenger RNA (mRNA), ribosomal RNA (rRNA) or transfer RNA (tRNA), forexample. The RNA may be a transcript. The RNA may be small RNA that areless than 200 nucleic acid bases in length, or large RNA that aregreater than 200 nucleic acid bases in length. Small RNAs may include5.8S ribosomal RNA (rRNA), 5S rRNA, transfer RNA (tRNA), microRNA(miRNA), small interfering RNA (siRNA), small nucleolar RNA (snoRNAs),Piwi-interacting RNA (piRNA), tRNA-derived small RNA (tsRNA) and smallrDNA-derived RNA (srRNA). The RNA may be double-stranded RNA orsingle-stranded RNA. The RNA may be circular RNA The macromolecularconstituent may comprise a protein. The macromolecular constituent maycomprise a peptide. The macromolecular constituent may comprise apolypeptide.

The term “molecular tag,” as used herein, generally refers to a moleculecapable of binding to a macromolecular constituent. The molecular tagmay bind to the macromolecular constituent with high affinity. Themolecular tag may bind to the macromolecular constituent with highspecificity. The molecular tag may comprise a nucleotide sequence. Themolecular tag may comprise a nucleic acid sequence. The nucleic acidsequence may be at least a portion or an entirety of the molecular tag.The molecular tag may be a nucleic acid molecule or may be part of anucleic acid molecule. The molecular tag may be an oligonucleotide or apolypeptide. The molecular tag may comprise a DNA aptamer. The moleculartag may be or comprise a primer. The molecular tag may be, or comprise,a protein. The molecular tag may comprise a polypeptide. The moleculartag may be a barcode.

The term “partition,” as used herein, generally, refers to a space orvolume that may be suitable to contain one or more species or conductone or more reactions. A partition may be a physical compartment, suchas a droplet or well. The partition may isolate space or volume fromanother space or volume. The droplet may be a first phase (e.g., aqueousphase) in a second phase (e.g., oil) immiscible with the first phase.The droplet may be a first phase in a second phase that does not phaseseparate from the first phase, such as, for example, a capsule orliposome in an aqueous phase. A partition may comprise one or more other(inner) partitions. In some cases, a partition may be a virtualcompartment that can be defined and identified by an index (e.g.,indexed libraries) across multiple and/or remote physical compartments.For example, a physical compartment may comprise a plurality of virtualcompartments.

The efficiency of many single cell applications can increase byimproving cell throughput. For example, this can be achieved by sortinga plurality of droplets that may or may not contain cells and/orparticles therein to collect only the droplets that contain the cellsand/or particles therein. The plurality of droplets may be sorted toisolate singularly occupied droplets from non-singularly occupieddroplets (e.g., unoccupied, multiply occupied, etc.). In anotherexample, higher efficiency can be achieved by isolating a plurality ofcell beads from a plurality of particles that may or may not containcells therein. The plurality of particles may be sorted to isolatesingularly occupied cell beads (e.g., particles containing cells ortheir derivatives) from non-singularly occupied cell beads (e.g.,unoccupied particles, multiply occupied cell beads, etc.). The isolatedpopulation of droplets that contain (e.g., singularly contain) the cellsand/or particles therein, and/or cell beads that contain (e.g.,singularly contain) the cells therein, can then be subject to furtherapplications, such as nucleic acid amplification and/or sequencingapplications.

Provided are methods and systems for sorting droplets. The methods andsystems generally operate by generating a plurality of droplets suchthat each of the plurality of droplets comprises field-attractableparticles. A given droplet in the plurality of droplets may or may notinclude therein one or more cells and/or other particles (e.g., cellbeads, gel beads, etc.). In some cases, the other particles (e.g., gelbeads) may have molecular barcodes coupled thereto. Thus, the pluralityof droplets comprising field attractable particles can comprise a firstsubset of droplets that include one or more cells and/or other particlesand a second subset of droplets that do not include any cells and/orother particles. A given droplet in the first subset of droplets thatincludes one or more cells and/or other particles can comprise asufficiently discrepant number or concentration of field-attractableparticles than a given droplet in the second subset of droplets thatdoes not include any cells and/or other particles such that when theplurality of droplets is subject to an electric or magnetic field, thefirst subset of droplets and the second subset of droplets are separatedfrom each other. In some cases, when the plurality of droplets issubjected to an electric or magnetic field, singularly occupied dropletsmay be separated from unoccupied droplets and otherwise multiplyoccupied droplets.

In some instances, a plurality of droplets can be generated with orwithout field-attractable particles. A given droplet in the plurality ofdroplets may or may not include one or more cells and/or particles.Thus, the plurality of droplets can comprise a first subset of dropletsthat include one or more cells and/or particles and a second subset ofdroplets that do not include any cells and/or particles. The pluralityof droplets can be subject to a pressure pulse and the first subset ofdroplets and the second subset of droplets can be separated from eachother via hydrodynamic forces. In some cases, singularly occupieddroplets may be separated from unoccupied droplets and otherwisemultiply occupied droplets.

In an aspect, the methods and systems described herein provide for thecompartmentalization, depositing, or partitioning of macromolecularconstituent contents of individual biological particles from a samplematerial containing biological particles into discrete compartments orpartitions (referred to interchangeably herein as partitions), whereeach partition maintains separation of its own contents from thecontents of other partitions. The partition can be a droplet in anemulsion. The partition can be a well. The partition can be a bead, suchas a gel bead and/or a cell bead. A partition may or may not containbiological particles and/or macromolecular constituents thereof. Inaccordance with some embodiments, each partition may contain at leastsome field attractable particles. The amount and/or concentration offield attractable particles in each partition can vary depending onwhether the partition contains biological particles (or other particles,such as beads). In accordance with some other embodiments, a partitionmay not contain field attractable particles.

In some instances, unique identifiers, such as barcodes, may bepreviously, subsequently or concurrently delivered to the partitionsthat hold the compartmentalized or partitioned biological particle, inorder to allow for the later attribution of the characteristics of theindividual biological particle to the particular partition. Barcodes maybe delivered, for example on an oligonucleotide, to a partition via anysuitable mechanism. Barcoded oligonucleotides can be delivered to apartition via a microcapsule. In some cases, barcoded oligonucleotidescan be initially associated with the microcapsule and then released fromthe microcapsule upon application of a stimulus which allows theoligonucleotides to dissociate or to be released from the microcapsule.

A microcapsule, in some instances, can comprise a bead. In some cases, abead may be porous, non-porous, solid, semi-solid, semi-fluidic,fluidic, and/or a combination thereof. In some instances, a bead may bedissolvable, disruptable, and/or degradable. In some cases, a bead maynot be degradable. In some cases, the bead may be a gel bead. A gel beadmay be a hydrogel bead. A gel bead may be formed from molecularprecursors, such as a polymeric or monomeric species. A semi-solid beadmay be a liposomal bead. Solid beads may comprise metals including ironoxide, gold, and silver. In some cases, the bead may be a silica bead.In some cases, the bead can be rigid. In other cases, the bead may beflexible and/or compressible.

In some instances, the bead may contain molecular precursors (e.g.,monomers or polymers), which may form a polymer network viapolymerization of the precursors. In some cases, a precursor may be analready polymerized species capable of undergoing further polymerizationvia, for example, a chemical cross-linkage. In some cases, a precursorcan comprise one or more of an acrylamide or a methacrylamide monomer,oligomer, or polymer. In some cases, the bead may comprise prepolymers,which are oligomers capable of further polymerization. For example,polyurethane beads may be prepared using prepolymers. In some cases, thebead may contain individual polymers that may be further polymerizedtogether. In some cases, beads may be generated via polymerization ofdifferent precursors, such that they comprise mixed polymers,co-polymers, and/or block co-polymers.

A bead may comprise natural and/or synthetic materials. For example, apolymer can be a natural polymer or a synthetic polymer. In some cases,a bead can comprise both natural and synthetic polymers. Examples ofnatural polymers include proteins and sugars such as deoxyribonucleicacid, rubber, cellulose, starch (e.g., amylose, amylopectin), proteins,enzymes, polysaccharides, silks, polyhydroxyalkanoates, chitosan,dextran, collagen, carrageenan, ispaghula, acacia, agar, gelatin,shellac, sterculia gum, xanthan gum, Corn sugar gum, guar gum, gumkaraya, agarose, alginic acid, alginate, or natural polymers thereof.Examples of synthetic polymers include acrylics, nylons, silicones,spandex, viscose rayon, polycarboxylic acids, polyvinyl acetate,polyacrylamide, polyacrylate, polyethylene glycol, polyurethanes,polylactic acid, silica, polystyrene, polyacrylonitrile, polybutadiene,polycarbonate, polyethylene, polyethylene terephthalate,poly(chlorotrifluoroethylene), poly(ethylene oxide), poly(ethyleneterephthalate), polyethylene, polyisobutylene, poly(methylmethacrylate), poly(oxymethylene), polyformaldehyde, polypropylene,polystyrene, poly(tetrafluoroethylene), poly(vinyl acetate), poly(vinylalcohol), poly(vinyl chloride), poly(vinylidene dichloride),poly(vinylidene difluoride), poly(vinyl fluoride) and/or combinations(e.g., co-polymers) thereof. Beads may also be formed from materialsother than polymers, including lipids, micelles, ceramics,glass-ceramics, material composites, metals, other inorganic materials,and others.

In some cases, a chemical cross-linker may be a precursor used tocross-link monomers during polymerization of the monomers and/or may beused to attach oligonucleotides (e.g., barcoded oligonucleotides) to thebead. In some cases, polymers may be further polymerized with across-linker species or other type of monomer to generate a furtherpolymeric network. Non-limiting examples of chemical cross-linkers (alsoreferred to as a “crosslinker” or a “crosslinker agent” herein) includecystamine, gluteraldehyde, dimethyl suberimidate, N-Hydroxysuccinimidecrosslinker BS3, formaldehyde, carbodiimide (EDC), SMCC, Sulfo-SMCC,vinylsilane, N,N′diallyltartardiamide (DATD),N,N′-Bis(acryloyl)cystamine (BAC), or homologs thereof. In some cases,the crosslinker used in the present disclosure contains cystamine.

Crosslinking may be permanent or reversible, depending upon theparticular crosslinker used. Reversible crosslinking may allow for thepolymer to linearize or dissociate under appropriate conditions. In somecases, reversible cross-linking may also allow for reversible attachmentof a material bound to the surface of a bead. In some cases, across-linker may form disulfide linkages. In some cases, the chemicalcross-linker forming disulfide linkages may be cystamine or a modifiedcystamine.

In some cases, disulfide linkages can be formed between molecularprecursor units (e.g., monomers, oligomers, or linear polymers) orprecursors incorporated into a bead and oligonucleotides. Cystamine(including modified cystamines), for example, is an organic agentcomprising a disulfide bond that may be used as a crosslinker agentbetween individual monomeric or polymeric precursors of a bead.Polyacrylamide may be polymerized in the presence of cystamine or aspecies comprising cystamine (e.g., a modified cystamine) to generatepolyacrylamide gel beads comprising disulfide linkages (e.g., chemicallydegradable beads comprising chemically-reducible cross-linkers). Thedisulfide linkages may permit the bead to be degraded (or dissolved)upon exposure of the bead to a reducing agent.

In some cases, chitosan, a linear polysaccharide polymer, may becrosslinked with glutaraldehyde via hydrophilic chains to form a bead.Crosslinking of chitosan polymers may be achieved by chemical reactionsthat are initiated by heat, pressure, change in pH, and/or radiation.

In some cases, the bead may comprise covalent or ionic bonds betweenpolymeric precursors (e.g., monomers, oligomers, linear polymers),oligonucleotides, primers, and other entities. In some cases, thecovalent bonds can be carbon-carbon bonds or thioether bonds.

In some cases, a bead may comprise an acrydite moiety, which in certainaspects may be used to attach one or more oligonucleotides (e.g.,barcode sequence, barcoded oligonucleotide, primer, or otheroligonucleotide) to the bead. In some cases, an acrydite moiety canrefer to an acrydite analogue generated from the reaction of acryditewith one or more species, such as, the reaction of acrydite with othermonomers and cross-linkers during a polymerization reaction. Acryditemoieties may be modified to form chemical bonds with a species to beattached, such as an oligonucleotide (e.g., barcode sequence, barcodedoligonucleotide, primer, or other oligonucleotide). Acrydite moietiesmay be modified with thiol groups capable of forming a disulfide bond ormay be modified with groups already comprising a disulfide bond. Thethiol or disulfide (via disulfide exchange) may be used as an anchorpoint for a species to be attached or another part of the acryditemoiety may be used for attachment. In some cases, attachment can bereversible, such that when the disulfide bond is broken (e.g., in thepresence of a reducing agent), the attached species is released from thebead. In other cases, an acrydite moiety can comprise a reactivehydroxyl group that may be used for attachment.

Functionalization of beads for attachment of oligonucleotides may beachieved through a wide range of different approaches, includingactivation of chemical groups within a polymer, incorporation of activeor activatable functional groups in the polymer structure, or attachmentat the pre-polymer or monomer stage in bead production.

For example, precursors (e.g., monomers, cross-linkers) that arepolymerized to form a bead may comprise acrydite moieties, such thatwhen a bead is generated, the bead also comprises acrydite moieties. Theacrydite moieties can be attached to a nucleic acid molecule (e.g.,oligonucleotide), which may include a priming sequence (e.g., a primerfor amplifying target nucleic acids, random primer, primer sequence formessenger RNA) and/or one or more barcode sequences. The one morebarcode sequences may include sequences that are the same for allnucleic acid molecules coupled to a given bead and/or sequences that aredifferent across all nucleic acid molecules coupled to the given bead.The nucleic acid molecule may be incorporated into the bead.

In some cases, the nucleic acid molecule can comprise a functionalsequence, for example, for attachment to a sequencing flow cell, suchas, for example, a P5 sequence for Illumina® sequencing. In some cases,the nucleic acid molecule or derivative thereof (e.g., oligonucleotideor polynucleotide generated from the nucleic acid molecule) can compriseanother functional sequence, such as, for example, a P7 sequence forattachment to a sequencing flow cell for Illumina® sequencing. In somecases, the nucleic acid molecule can comprise a barcode sequence. Insome cases, the primer can further comprise a unique molecularidentifier (UMI). In some cases, the primer can comprise an R1 primersequence for Illumina sequencing. In some cases, the primer can comprisean R2 primer sequence for Illumina sequencing. Examples of such nucleicacid molecules (e.g., oligonucleotides, polynucleotides, etc.) and usesthereof, as may be used with compositions, devices, methods and systemsof the present disclosure, are provided in U.S. Patent Pub. Nos.2014/0378345 and 2015/0376609, each of which is entirely incorporatedherein by reference.

FIG. 19 illustrates an example of a barcode carrying bead. A nucleicacid molecule 1902, such as an oligonucleotide, can be coupled to a bead1904 by a releasable linkage 1906, such as, for example, a disulfidelinker. The same bead 1904 may be coupled (e.g., via releasable linkage)to one or more other nucleic acid molecules 1918, 1920. The nucleic acidmolecule 1902 may be or comprise a barcode. As noted elsewhere herein,the structure of the barcode may comprise a number of sequence elements.The nucleic acid molecule 1902 may comprise a functional sequence 1908that may be used in subsequent processing. For example, the functionalsequence 1908 may include one or more of a sequencer specific flow cellattachment sequence (e.g., a P5 sequence for Illumina® sequencingsystems) and a sequencing primer sequence (e.g., a R1 primer forIllumina® sequencing systems). The nucleic acid molecule 1902 maycomprise a barcode sequence 1910 for use in barcoding the sample (e.g.,DNA, RNA, protein, etc.). In some cases, the barcode sequence 1910 canbe bead-specific such that the barcode sequence 1910 is common to allnucleic acid molecules (e.g., including nucleic acid molecule 1902)coupled to the same bead 1904. Alternatively or in addition, the barcodesequence 1910 can be partition-specific such that the barcode sequence1910 is common to all nucleic acid molecules coupled to one or morebeads that are partitioned into the same partition. The nucleic acidmolecule 1902 may comprise a specific priming sequence 1912, such as anmRNA specific priming sequence (e.g., poly-T sequence), a targetedpriming sequence, and/or a random priming sequence. The nucleic acidmolecule 1902 may comprise an anchoring sequence 1914 to ensure that thespecific priming sequence 1912 hybridizes at the sequence end (e.g., ofthe mRNA). For example, the anchoring sequence 1914 can include a randomshort sequence of nucleotides, such as a 1-mer, 2-mer, 3-mer or longersequence, which can ensure that a poly-T segment is more likely tohybridize at the sequence end of the poly-A tail of the mRNA.

The nucleic acid molecule 1902 may comprise a unique molecularidentifying sequence 1916 (e.g., unique molecular identifier (UMI)). Insome cases, the unique molecular identifying sequence 1916 may comprisefrom about 5 to about 8 nucleotides. Alternatively, the unique molecularidentifying sequence 1916 may compress less than about 5 or more thanabout 8 nucleotides. The unique molecular identifying sequence 1916 maybe a unique sequence that varies across individual nucleic acidmolecules (e.g., 1902, 1918, 1920, etc.) coupled to a single bead (e.g.,bead 1904). In some cases, the unique molecular identifying sequence1916 may be a random sequence (e.g., such as a random N-mer sequence).For example, the UMI may provide a unique identifier of the startingmRNA molecule that was captured, in order to allow quantitation of thenumber of original expressed RNA. As will be appreciated, although FIG.19 shows three nucleic acid molecules 1902, 1918, 1920 coupled to thesurface of the bead 1904, an individual bead may be coupled to anynumber of individual nucleic acid molecules, for example, from one totens to hundreds of thousands or even millions of individual nucleicacid molecules. The respective barcodes for the individual nucleic acidmolecules can comprise both common sequence segments or relativelycommon sequence segments (e.g., 1908, 1910, 1912, etc.) and variable orunique sequence segments (e.g., 1916) between different individualnucleic acid molecules coupled to the same bead.

In operation, a biological particle (e.g., cell, DNA, RNA, etc.) can beco-partitioned along with a barcode bearing bead 1904. The barcodednucleic acid molecules 1902, 1918, 1920 can be released from the bead1904 in the partition. By way of example, in the context of analyzingsample RNA, the poly-T segment (e.g., 1912) of one of the releasednucleic acid molecules (e.g., 1902) can hybridize to the poly-A tail ofa mRNA molecule. Reverse transcription may result in a cDNA transcriptof the mRNA, but which transcript includes each of the sequence segments1908, 1910, 1916 of the nucleic acid molecule 1902. Because the nucleicacid molecule 1902 comprises an anchoring sequence 1914, it will morelikely hybridize to and prime reverse transcription at the sequence endof the poly-A tail of the mRNA. Within any given partition, all of thecDNA transcripts of the individual mRNA molecules may include a commonbarcode sequence segment 1910. However, the transcripts made from thedifferent mRNA molecules within a given partition may vary at the uniquemolecular identifying sequence 1912 segment (e.g., UMI segment).Beneficially, even following any subsequent amplification of thecontents of a given partition, the number of different UMIs can beindicative of the quantity of mRNA originating from a given partition,and thus from the biological particle (e.g., cell). As noted above, thetranscripts can be amplified, cleaned up and sequenced to identify thesequence of the cDNA transcript of the mRNA, as well as to sequence thebarcode segment and the UMI segment. While a poly-T primer sequence isdescribed, other targeted or random priming sequences may also be usedin priming the reverse transcription reaction. Likewise, althoughdescribed as releasing the barcoded oligonucleotides into the partition,in some cases, the nucleic acid molecules bound to the bead (e.g., gelbead) may be used to hybridize and capture the mRNA on the solid phaseof the bead, for example, in order to facilitate the separation of theRNA from other cell contents.

In some cases, precursors comprising a functional group that is reactiveor capable of being activated such that it becomes reactive can bepolymerized with other precursors to generate gel beads comprising theactivated or activatable functional group. The functional group may thenbe used to attach additional species (e.g., disulfide linkers, primers,other oligonucleotides, etc.) to the gel beads. For example, someprecursors comprising a carboxylic acid (COOH) group can co-polymerizewith other precursors to form a gel bead that also comprises a COOHfunctional group. In some cases, acrylic acid (a species comprising freeCOOH groups), acrylamide, and bis(acryloyl)cystamine can beco-polymerized together to generate a gel bead comprising free COOHgroups. The COOH groups of the gel bead can be activated (e.g., via1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) andN-Hydroxysuccinimide (NHS) or4-(4,6-Dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride(DMTMM)) such that they are reactive (e.g., reactive to amine functionalgroups where EDC/NHS or DMTMM are used for activation). The activatedCOOH groups can then react with an appropriate species (e.g., a speciescomprising an amine functional group where the carboxylic acid groupsare activated to be reactive with an amine functional group) comprisinga moiety to be linked to the bead.

Beads comprising disulfide linkages in their polymeric network may befunctionalized with additional species via reduction of some of thedisulfide linkages to free thiols. The disulfide linkages may be reducedvia, for example, the action of a reducing agent (e.g., DTT, TCEP, etc.)to generate free thiol groups, without dissolution of the bead. Freethiols of the beads can then react with free thiols of a species or aspecies comprising another disulfide bond (e.g., via thiol-disulfideexchange) such that the species can be linked to the beads (e.g., via agenerated disulfide bond). In some cases, free thiols of the beads mayreact with any other suitable group. For example, free thiols of thebeads may react with species comprising an acrydite moiety. The freethiol groups of the beads can react with the acrydite via Michaeladdition chemistry, such that the species comprising the acrydite islinked to the bead. In some cases, uncontrolled reactions can beprevented by inclusion of a thiol capping agent such asN-ethylmalieamide or iodoacetate.

Activation of disulfide linkages within a bead can be controlled suchthat only a small number of disulfide linkages are activated. Controlmay be exerted, for example, by controlling the concentration of areducing agent used to generate free thiol groups and/or concentrationof reagents used to form disulfide bonds in bead polymerization. In somecases, a low concentration (e.g., molecules of reducing agent:gel beadratios of less than or equal to about 1:100,000,000,000, less than orequal to about 1:10,000,000,000, less than or equal to about1:1,000,000,000, less than or equal to about 1:100,000,000, less than orequal to about 1:10,000,000, less than or equal to about 1:1,000,000,less than or equal to about 1:100,000, less than or equal to about1:10,000) of reducing agent may be used for reduction. Controlling thenumber of disulfide linkages that are reduced to free thiols may beuseful in ensuring bead structural integrity during functionalization.In some cases, optically-active agents, such as fluorescent dyes may becoupled to beads via free thiol groups of the beads and used to quantifythe number of free thiols present in a bead and/or track a bead.

In some cases, addition of moieties to a gel bead after gel beadformation may be advantageous. For example, addition of anoligonucleotide (e.g., barcoded oligonucleotide) after gel beadformation may avoid loss of the species during chain transfertermination that can occur during polymerization. Moreover, smallerprecursors (e.g., monomers or cross linkers that do not comprise sidechain groups and linked moieties) may be used for polymerization and canbe minimally hindered from growing chain ends due to viscous effects. Insome cases, functionalization after gel bead synthesis can minimizeexposure of species (e.g., oligonucleotides) to be loaded withpotentially damaging agents (e.g., free radicals) and/or chemicalenvironments. In some cases, the generated gel may possess an uppercritical solution temperature (UCST) that can permit temperature drivenswelling and collapse of a bead. Such functionality may aid inoligonucleotide (e.g., a primer) infiltration into the bead duringsubsequent functionalization of the bead with the oligonucleotide.Post-production functionalization may also be useful in controllingloading ratios of species in beads, such that, for example, thevariability in loading ratio is minimized. Species loading may also beperformed in a batch process such that a plurality of beads can befunctionalized with the species in a single batch.

In some cases, beads can be non-covalently loaded with one or morereagents. The beads can be non-covalently loaded by, for instance,subjecting the beads to conditions sufficient to swell the beads,allowing sufficient time for the reagents to diffuse into the interiorsof the beads, and subjecting the beads to conditions sufficient tode-swell the beads. The swelling of the beads may be accomplished, forinstance, by placing the beads in a thermodynamically favorable solvent,subjecting the beads to a higher or lower temperature, subjecting thebeads to a higher or lower ion concentration, and/or subjecting thebeads to an electric field. The swelling of the beads may beaccomplished by any swelling method as is known to one having skill inthe art. The de-swelling of the beads may be accomplished, for instance,by transferring the beads in a thermodynamically unfavorable solvent,subjecting the beads to lower or high temperatures, subjecting the beadsto a lower or higher ion concentration, and/or removing an electricfield. The de-swelling of the beads may be accomplished by anyde-swelling method as is known to one having skill in the art.Transferring the beads may cause pores in the bead to shrink. Theshrinking may then hinder reagents within the beads from diffusing outof the interiors of the beads. The hindrance may be due to stericinteractions between the reagents and the interiors of the beads. Thetransfer may be accomplished microfluidically. For instance, thetransfer may be achieved by moving the beads from one co-flowing solventstream to a different co-flowing solvent stream. The swellability and/orpore size of the beads may be adjusted by changing the polymercomposition of the bead.

In some cases, an acrydite moiety linked to precursor, another specieslinked to a precursor, or a precursor itself comprises a labile bond,such as chemically, thermally, or photo-sensitive bonds e.g., disulfidebonds, UV sensitive bonds, or the like. Once acrydite moieties or othermoieties comprising a labile bond are incorporated into a bead, the beadmay also comprise the labile bond. The labile bond may be, for example,useful in reversibly linking (e.g., covalently linking) species (e.g.,barcodes, primers, etc.) to a bead. In some cases, a thermally labilebond may include a nucleic acid hybridization based attachment, e.g.,where an oligonucleotide is hybridized to a complementary sequence thatis attached to the bead, such that thermal melting of the hybridreleases the oligonucleotide, e.g., a barcode containing sequence, fromthe bead or microcapsule.

The addition of multiple types of labile bonds to a gel bead may resultin the generation of a bead capable of responding to varied stimuli.Each type of labile bond may be sensitive to an associated stimulus(e.g., chemical stimulus, light, temperature, etc.) such that release ofspecies attached to a bead via each labile bond may be controlled by theapplication of the appropriate stimulus. Such functionality may beuseful in controlled release of species from a gel bead. In some cases,another species comprising a labile bond may be linked to a gel beadafter gel bead formation via, for example, an activated functional groupof the gel bead as described above. As will be appreciated, barcodesthat are releasably, cleavably or reversibly attached to the beadsdescribed herein include barcodes that are released or releasablethrough cleavage of a linkage between the barcode molecule and the bead,or that are released through degradation of the underlying bead itself,allowing the barcodes to be accessed or accessible by other reagents, orboth.

The barcodes that are releasable as described herein may sometimes bereferred to as being activatable, in that they are available forreaction once released. Thus, for example, an activatable barcode may beactivated by releasing the barcode from a bead (or other suitable typeof partition described herein). Other activatable configurations arealso envisioned in the context of the described methods and systems.

In addition to thermally cleavable bonds, disulfide bonds and UVsensitive bonds, other non-limiting examples of labile bonds that may becoupled to a precursor or bead include an ester linkage (e.g., cleavablewith an acid, a base, or hydroxylamine), a vicinal diol linkage (e.g.,cleavable via sodium periodate), a Diels-Alder linkage (e.g., cleavablevia heat), a sulfone linkage (e.g., cleavable via a base), a silyl etherlinkage (e.g., cleavable via an acid), a glycosidic linkage (e.g.,cleavable via an amylase), a peptide linkage (e.g., cleavable via aprotease), or a phosphodiester linkage (e.g., cleavable via a nuclease(e.g., DNAase)).

Species that do not participate in polymerization may also beencapsulated in beads during bead generation (e.g., duringpolymerization of precursors). Such species may be entered intopolymerization reaction mixtures such that generated beads comprise thespecies upon bead formation. In some cases, such species may be added tothe gel beads after formation. Such species may include, for example,oligonucleotides, reagents for a nucleic acid amplification reaction(e.g., primers, polymerases, dNTPs, co-factors (e.g., ionic co-factors))including those described herein, reagents for enzymatic reactions(e.g., enzymes, co-factors, substrates), or reagents for a nucleic acidmodification reactions such as polymerization, ligation, or digestion.Trapping of such species may be controlled by the polymer networkdensity generated during polymerization of precursors, control of ioniccharge within the gel bead (e.g., via ionic species linked topolymerized species), or by the release of other species. Encapsulatedspecies may be released from a bead upon bead degradation and/or byapplication of a stimulus capable of releasing the species from thebead.

Beads may be of uniform size or heterogeneous size. In some cases, thediameter of a bead may be at least about 1 micrometers (μm), 90 μm, 100μm, 250 μm, 500 μm, 1 mm, or greater. In some cases, a bead may have adiameter of less than about 90 μm, 100 μm, 250 μm, 500 μm, 1 mm, orless. In some cases, a bead may have a diameter in the range of about40-75 μm, 30-75 μm, 20-75 μm, 40-85 μm, 40-95 μm, 20-100 μm, 10-100 μm,1-100 μm, 20-250 μm, or 20-500 μm.

In certain aspects, beads can be provided as a population or pluralityof beads having a relatively monodisperse size distribution. Where itmay be desirable to provide relatively consistent amounts of reagentswithin partitions, maintaining relatively consistent beadcharacteristics, such as size, can contribute to the overallconsistency. In particular, the beads described herein may have sizedistributions that have a coefficient of variation in theircross-sectional dimensions of less than 50%, less than 40%, less than30%, less than 20%, and in some cases less than 15%, less than 10%, lessthan 5%, or less.

Beads may be of any suitable shape. Examples of bead shapes include, butare not limited to, spherical, non-spherical, oval, oblong, amorphous,circular, cylindrical, and variations thereof.

In addition to, or as an alternative to the cleavable linkages betweenthe beads and the associated molecules, such as barcode containingoligonucleotides, described above, the beads may be degradable,disruptable, or dissolvable spontaneously or upon exposure to one ormore stimuli (e.g., temperature changes, pH changes, exposure toparticular chemical species or phase, exposure to light, reducing agent,etc.). In some cases, a bead may be dissolvable, such that materialcomponents of the beads are solubilized when exposed to a particularchemical species or an environmental change, such as a changetemperature or a change in pH. In some cases, a gel bead can be degradedor dissolved at elevated temperature and/or in basic conditions. In somecases, a bead may be thermally degradable such that when the bead isexposed to an appropriate change in temperature (e.g., heat), the beaddegrades. Degradation or dissolution of a bead bound to a species (e.g.,a oligonucleotide, e.g., barcoded oligonucleotide) may result in releaseof the species from the bead.

A degradable bead may comprise one or more species with a labile bondsuch that, when the bead/species is exposed to the appropriate stimuli,the bond is broken and the bead degrades. The labile bond may be achemical bond (e.g., covalent bond, ionic bond) or may be another typeof physical interaction (e.g., van der Waals interactions, dipole-dipoleinteractions, etc.). In some cases, a crosslinker used to generate abead may comprise a labile bond. Upon exposure to the appropriateconditions, the labile bond can be broken and the bead degraded. Forexample, upon exposure of a polyacrylamide gel bead comprising cystaminecrosslinkers to a reducing agent, the disulfide bonds of the cystaminecan be broken and the bead degraded.

A degradable bead may be useful in more quickly releasing an attachedspecies (e.g., an oligonucleotide, a barcode sequence, a primer, etc)from the bead when the appropriate stimulus is applied to the bead ascompared to a bead that does not degrade. For example, for a speciesbound to an inner surface of a porous bead or in the case of anencapsulated species, the species may have greater mobility andaccessibility to other species in solution upon degradation of the bead.In some cases, a species may also be attached to a degradable bead via adegradable linker (e.g., disulfide linker). The degradable linker mayrespond to the same stimuli as the degradable bead or the two degradablespecies may respond to different stimuli. For example, a barcodesequence may be attached, via a disulfide bond, to a polyacrylamide beadcomprising cystamine. Upon exposure of the barcoded-bead to a reducingagent, the bead degrades and the barcode sequence is released uponbreakage of both the disulfide linkage between the barcode sequence andthe bead and the disulfide linkages of the cystamine in the bead.

A degradable bead may be introduced into a partition, such as a dropletof an emulsion or a well, such that the bead degrades within thepartition and any associated species (e.g., oligonucleotides) arereleased within the droplet when the appropriate stimulus is applied.The free species (e.g., oligonucleotides) may interact with otherreagents contained in the partition. For example, a polyacrylamide beadcomprising cystamine and linked, via a disulfide bond, to a barcodesequence, may be combined with a reducing agent within a droplet of awater-in-oil emulsion. Within the droplet, the reducing agent breaks thevarious disulfide bonds resulting in bead degradation and release of thebarcode sequence into the aqueous, inner environment of the droplet. Inanother example, heating of a droplet comprising a bead-bound barcodesequence in basic solution may also result in bead degradation andrelease of the attached barcode sequence into the aqueous, innerenvironment of the droplet.

As will be appreciated from the above disclosure, while referred to asdegradation of a bead, in many instances as noted above, thatdegradation may refer to the disassociation of a bound or entrainedspecies from a bead, both with and without structurally degrading thephysical bead itself. For example, entrained species may be releasedfrom beads through osmotic pressure differences due to, for example,changing chemical environments. By way of example, alteration of beadpore sizes due to osmotic pressure differences can generally occurwithout structural degradation of the bead itself. In some cases, anincrease in pore size due to osmotic swelling of a bead can permit therelease of entrained species within the bead. In other cases, osmoticshrinking of a bead may cause a bead to better retain an entrainedspecies due to pore size contraction.

Where degradable beads are provided, it may be desirable to avoidexposing such beads to the stimulus or stimuli that cause suchdegradation prior to the desired time, in order to avoid premature beaddegradation and issues that arise from such degradation, including forexample poor flow characteristics and aggregation. By way of example,where beads comprise reducible cross-linking groups, such as disulfidegroups, it will be desirable to avoid contacting such beads withreducing agents, e.g., DTT or other disulfide cleaving reagents. In suchcases, treatment to the beads described herein will, in some cases beprovided free of reducing agents, such as DTT. Because reducing agentsare often provided in commercial enzyme preparations, it may bedesirable to provide reducing agent free (or DTT free) enzymepreparations in treating the beads described herein. Examples of suchenzymes include, e.g., polymerase enzyme preparations, reversetranscriptase enzyme preparations, ligase enzyme preparations, as wellas many other enzyme preparations that may be used to treat the beadsdescribed herein. The terms “reducing agent free” or “DTT free”preparations can refer to a preparation having less than about 1/10th,less than about 1/50th, or even less than about 1/100th of the lowerranges for such materials used in degrading the beads. For example, forDTT, the reducing agent free preparation can have less than about 0.01millimolar (mM), 0.005 mM, 0.001 mM DTT, 0.0005 mM DTT, or even lessthan about 0.0001 mM DTT. In many cases, the amount of DTT can beundetectable.

Numerous chemical triggers may be used to trigger the degradation ofbeads. Examples of these chemical changes may include, but are notlimited to pH-mediated changes to the integrity of a component withinthe bead, degradation of a component of a bead via cleavage ofcross-linked bonds, and depolymerization of a component of a bead.

In some embodiments, a bead may be formed from materials that comprisedegradable chemical crosslinkers, such as BAC or cystamine. Degradationof such degradable crosslinkers may be accomplished through a number ofmechanisms. In some examples, a bead may be contacted with a chemicaldegrading agent that may induce oxidation, reduction or other chemicalchanges. For example, a chemical degrading agent may be a reducingagent, such as dithiothreitol (DTT). Additional examples of reducingagents may include β-mercaptoethanol, (2S)-2-amino-1,4-dimercaptobutane(dithiobutylamine or DTBA), tris(2-carboxyethyl) phosphine (TCEP), orcombinations thereof. A reducing agent may degrade the disulfide bondsformed between gel precursors forming the bead, and thus, degrade thebead. In other cases, a change in pH of a solution, such as an increasein pH, may trigger degradation of a bead. In other cases, exposure to anaqueous solution, such as water, may trigger hydrolytic degradation, andthus degradation of the bead.

Beads may also be induced to release their contents upon the applicationof a thermal stimulus. A change in temperature can cause a variety ofchanges to a bead. For example, heat can cause a solid bead to liquefy.A change in heat may cause melting of a bead such that a portion of thebead degrades. In other cases, heat may increase the internal pressureof the bead components such that the bead ruptures or explodes. Heat mayalso act upon heat-sensitive polymers used as materials to constructbeads.

The methods, compositions, devices, and kits of this disclosure may beused with any suitable agent to degrade beads. In some embodiments,changes in temperature or pH may be used to degrade thermo-sensitive orpH-sensitive bonds within beads. In some embodiments, chemical degradingagents may be used to degrade chemical bonds within beads by oxidation,reduction or other chemical changes. For example, a chemical degradingagent may be a reducing agent, such as DTT, wherein DTT may degrade thedisulfide bonds formed between a crosslinker and gel precursors, thusdegrading the bead. In some embodiments, a reducing agent may be addedto degrade the bead, which may or may not cause the bead to release itscontents. Examples of reducing agents may include dithiothreitol (DTT),β-mercaptoethanol, (2S)-2-amino-1,4-dimercaptobutane (dithiobutylamineor DTBA), tris(2-carboxyethyl) phosphine (TCEP), or combinationsthereof. The reducing agent may be present at a concentration of about0.1 mM, 0.5 mM, 1 mM, 5 mM, 10 mM. The reducing agent may be present ata concentration of at least about 0.1 mM, 0.5 mM, 1 mM, 5 mM, 10 mM, orgreater than 10 mM. The reducing agent may be present at concentrationof at most about 10 mM, 5 mM, 1 mM, 0.5 mM, 0.1 mM, or less.

Any suitable number of molecular tag molecules (e.g., primer, barcodedoligonucleotide) can be associated with a bead such that, upon releasefrom the bead, the molecular tag molecules (e.g., primer, e.g., barcodedoligonucleotide) are present in the partition at a pre-definedconcentration. Such pre-defined concentration may be selected tofacilitate certain reactions for generating a sequencing library, e.g.,amplification, within the partition. In some cases, the pre-definedconcentration of the primer can be limited by the process of producingoligonucleotide bearing beads.

The compartments or partitions can be flowable within fluid streams. Thepartitions may comprise, for example, micro-vesicles that have an outerbarrier surrounding an inner fluid center or core. In some cases, thepartitions may comprise a porous matrix that is capable of entrainingand/or retaining materials within its matrix. The partitions cancomprise droplets of aqueous fluid within a non-aqueous continuousphase, e.g., an oil phase. The partitions can comprise droplets of afirst phase within a second phase, wherein the first and second phasesare immiscible. A variety of different vessels are described in, forexample, U.S. Patent Application Publication No. 2014/0155295, which isentirely incorporated herein by reference for all purposes. Emulsionsystems for creating stable droplets in non-aqueous or oil continuousphases are described in detail in, e.g., U.S. Patent ApplicationPublication No. 2010/0105112, which is entirely incorporated herein byreference for all purposes.

In the case of droplets in an emulsion, allocating individual biologicalparticles to discrete partitions may generally be accomplished byintroducing a flowing stream of biological particles in an aqueous fluidinto a flowing stream of a non-aqueous fluid, such that droplets aregenerated at the junction of the two streams. By providing the aqueousstream at a certain concentration of biological particles, the occupancyof the resulting partitions (e.g., number of biological particles perpartition) can be controlled. Where single biological particlepartitions are desired, the relative flow rates of the immiscible fluidscan be selected such that, on average, the partitions contain less thanone biological particle per partition, in order to ensure that thosepartitions that are occupied, are primarily singularly occupied. In someembodiments, the relative flow rates of the fluids can be selected suchthat a majority of partitions are occupied, e.g., allowing for only asmall percentage of unoccupied partitions. The flows and channelarchitectures can be controlled as to ensure a desired number ofsingularly occupied partitions, less than a certain level of unoccupiedpartitions and/or less than a certain level of multiply occupiedpartitions.

The systems and methods described herein can be operated such that amajority of occupied partitions include no more than one biologicalparticle per occupied partition. In some cases, the partitioning processis conducted such that fewer than 25% of the occupied partitions containmore than one biological particle, and in many cases, fewer than 20% ofthe occupied partitions have more than one biological particle. In somecases, fewer than 10% or even fewer than 5% of the occupied partitionsinclude more than one biological particle per partition.

In some cases, it is desirable to avoid the creation of excessivenumbers of empty partitions. For example, from a cost perspective and/orefficiency perspective, it may desirable to minimize the number of emptypartitions. However, while this may be accomplished by providingsufficient numbers of biological particles into the partitioning zone,the Poissonian distribution may expectedly increase the number ofpartitions that may include multiple biological particles. As such, atmost about 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%,35%, 30%, 25%, 20%, 15%, 10%, 5% or less of the generated partitions canbe unoccupied. In some cases, the flow of one or more of the cells, orother fluids directed into the partitioning zone can be conducted suchthat, in many cases, no more than about 50% of the generated partitions,no more than about 25% of the generated partitions, or no more thanabout 10% of the generated partitions are unoccupied. These flows can becontrolled so as to present non-Poissonian distribution of singleoccupied partitions while providing lower levels of unoccupiedpartitions. The above noted ranges of unoccupied partitions can beachieved while still providing any of the single occupancy ratesdescribed above. For example, in many cases, the use of the systems andmethods described herein creates resulting partitions that have multipleoccupancy rates of less than about 25%, less than about 20%, less thanabout 15%, less than about 10%, and in many cases, less than about 5%,while having unoccupied partitions of less than about 50%, less thanabout 40%, less than about 30%, less than about 20%, less than about10%, less than about 5%, or less.

After the partitions are generated, comprising in part singularlyoccupied partitions, in part multiply occupied partitions, and/or inpart unoccupied partitions, the occupied partitions can be sorted fromthe unoccupied partitions. In some cases, singularly occupied partitionsmay be isolated from non-singularly occupied partitions (e.g., multiplyoccupied partitions and unoccupied partitions). Such sorting can beachieved by including field attractable particles during the generationof droplets in an emulsion. For example, a flowing stream of aqueousfluid containing biological particles and field attractable particlescan be introduced into a flowing stream of a non-aqueous fluid, suchthat droplets are generated at the junction of the two streams.

As will be appreciated, the above-described occupancy rates are alsoapplicable to partitions that include both biological particles andadditional reagents, including, but not limited to, microcapsulescarrying barcoded oligonucleotides. The occupied partitions (e.g., atleast about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% ofthe occupied partitions) can include both a microcapsule (e.g., bead)comprising barcoded oligonucleotides and a biological particle.

Although described in terms of providing substantially singularlyoccupied partitions, above, in certain cases, it is desirable to providemultiply occupied partitions, e.g., containing two, three, four or morecells and/or microcapsules (e.g., beads) comprising barcodedoligonucleotides within a single partition. Accordingly, as noted above,the flow characteristics of the biological particle and/or beadcontaining fluids and partitioning fluids may be controlled to providefor such multiply occupied partitions. In particular, the flowparameters may be controlled to provide a desired occupancy rate atgreater than about 50% of the partitions, greater than about 75%, and insome cases greater than about 80%, 90%, 95%, or higher.

In some cases, additional microcapsules are used to deliver additionalreagents to a partition. In such cases, it may be advantageous tointroduce different beads into a common channel or droplet generationjunction, from different bead sources, i.e., containing differentassociated reagents, through different channel inlets into such commonchannel or droplet generation junction. In such cases, the flow andfrequency of the different beads into the channel or junction may becontrolled to provide for the desired ratio of microcapsules from eachsource, while ensuring the desired pairing or combination of such beadsinto a partition with the desired number of biological particles.

The partitions described herein may comprise small volumes, e.g., lessthan about 10 microliters (μL), 5 μL, 1 μL, 900 picoliters (μL), 800 μL,700 μL, 600 μL, 500 μL, 400 μL, 300 μL, 200 μL, 100 μL, 50 μL, 20 μL, 10μL, 1 μL, 500 nanoliters (nL), 100 nL, 50 nL, or less.

For example, in the case of droplet based partitions, the droplets mayhave overall volumes that are less than about 1000 μL, 900 μL, 800 μL,700 μL, 600 μL, 500 μL, 400 μL, 300 μL, 200 μL, 100 μL, 50 μL, 20 μL, 10μL, 1 μL, or less. Where co-partitioned with microcapsules, it will beappreciated that the sample fluid volume, e.g., including co-partitionedbiological particles, within the partitions may be less than about 90%of the above described volumes, less than about 80%, less than about70%, less than about 60%, less than about 50%, less than about 40%, lessthan about 30%, less than about 20%, or less than about 10% the abovedescribed volumes.

As is described elsewhere herein, partitioning species may generate apopulation or plurality of partitions. In such cases, any suitablenumber of partitions can be generated to generate the plurality ofpartitions. For example, in a method described herein, a plurality ofpartitions may be generated that comprises at least about 1,000partitions, at least about 5,000 partitions, at least about 10,000partitions, at least about 50,000 partitions, at least about 100,000partitions, at least about 500,000 partitions, at least about 1,000,000partitions, at least about 5,000,000 partitions at least about10,000,000 partitions, at least about 50,000,000 partitions, at leastabout 100,000,000 partitions, at least about 500,000,000 partitions, atleast about 1,000,000,000 partitions, or more. Moreover, the pluralityof partitions may comprise both unoccupied partitions (e.g., emptypartitions) and occupied partitions.

Microfluidic channel networks can be utilized to generate partitions asdescribed herein. Alternative mechanisms may also be employed in thepartitioning of individual biological particles, including porousmembranes through which aqueous mixtures of cells are extruded intonon-aqueous fluids.

FIG. 1 shows an example of a microfluidic channel structure forpartitioning individual biological particles. As described elsewhereherein, in some cases, the majority of occupied partitions can includeno more than one biological particle per occupied partition and, in somecases, some of the generated partitions can be unoccupied (of anybiological particle). In some cases, though, some of the occupiedpartitions may include more than one biological particle. In some cases,the partitioning process may be controlled such that fewer than about25% of the occupied partitions contain more than one biologicalparticle, and in many cases, fewer than about 20% of the occupiedpartitions have more than one biological particle, while in some cases,fewer than about 10% or even fewer than about 5% of the occupiedpartitions include more than one biological particle per partition.

As shown in FIG. 1, the channel structure can include channel segments102, 104, 106 and 108 communicating at a channel junction 110. Inoperation, a first aqueous fluid 112 that includes suspended biologicalparticles (e.g., cells) 114 and suspended field-attractable particles115, may be transported along channel segment 102 into junction 110,while a second fluid 116 that is immiscible with the aqueous fluid 112is delivered to the junction 110 from each of channel segments 104 and106 to create discrete droplets 118, 120 of the first aqueous fluid 112flowing into channel segment 108, and flowing away from junction 110. Adiscrete droplet generated may include an individual biological particle114 and field-attractable particles 115 (such as droplets 118). Adiscrete droplet generated may include more than one individualbiological particle 114 and field-attractable particles 115 (not shownin FIG. 1). A discrete droplet may contain field-attractable particles115 but no biological particle 114 (such as droplet 120).

The field-attractable particles 115 may be paramagnetic particles. Insome cases, the field-attractable particles may be superparamagneticparticles. For example, the field-attractable particles can comprisepolystyrene magnetic particles (e.g., polystyrene core particle coatedwith at least a layer of magnetite (e.g., iron oxide) and polystyrene),amino magnetic particles, carboxyl magnetic particles, dimethylaminomagnetic particles, hydroxyethyl magnetic particles, and/or acombination of the above. A paramagnetic particle can comprise a polymermatrix of amine silane, glucuronic acid, bromoacetyl, chitosan,carboxymethyldextran, citric acid, starch, DEAE-starch,phosphate-starch, dextran, dextran-sulfate, lipid, oleic acid,diphosphate, polyaspartic acid, polyacrylamide, polyacrylic acid,polydimethylamine, polyethylene glycole alpha-methoxy-omega-amine,polyethylene glycol alpha-,omega-diphosphate, poly(maleicacid-co-olefin), polystyrenesulfonate, polyvinyl alcohol,poly(4-vinylpyridine), poly-diallyldimethylamin, uncoated magnetite,and/or other matrices. A paramagnetic particle may have micrometer ornanometer size. For example, a paramagnetic particle can have a maximumdimension (e.g., width, length, height, diameter, etc.) of at most about20 micrometers (μm), 10 μm, 9 μm, 8 μm, 7 μm, 6 μm, 5 μm, 4 μm, 3 μm, 2μm, 1 μm, 0.5 μm, or less. The paramagnetic particle can have a maximumdimension of at most about 500 nanometer (nm), 400 nm, 300 nm, 200 nm,100 nm, 50 nm, 40 nm, 30 nm, 20 nm, 10 nm, 9 nm, 8 nm, 7 nm, 6 nm, 5 nm,4 nm, 3 nm, 2 nm, 1 nm, or less. Alternatively, a paramagnetic particlecan have a maximum dimension that is greater than about 20 μm. Theparamagnetic particles may be responsive when exposed to magneticfields. In some cases, the paramagnetic particles can be smooth surfaceparticles, wherein a thick polymer layer coats the magnetite (e.g., ironoxide) layer. The smooth surface can shield the magnetite frominterfering with enzyme activities or other undesirable effects withother particles or cells caused by exposure to the magnetite. In somecases, the paramagnetic particles can be cross-linked particles, whereinthe particles are coated with cross-linked polymer on the surfaces ofthe iron oxide crystals. The cross-linked polymer can render theparamagnetic particle resistant to common organic solvents, such asacetone, acetonitrile, dimethlyformanide (DMF) and chloroform. Themagnetite content on each paramagnetic particle can be adjusted (e.g.,to have higher or lower percentage) to be more responsive or lessresponsive to the same magnetic field. In some cases, thefield-attractable particles can be diamagnetic particles orferromagnetic particles.

In some cases, the field-attractable particles 115 may be conductiveparticles. A conductive particle may have micrometer or nanometer size.For example, a conductive particle can have a maximum dimension (e.g.,width, length, height, diameter, etc.) of at most about 20 micrometers(μm), 10 μm, 9 μm, 8 μm, 7 μm, 6 μm, 5 μm, 4 μm, 3 μm, 2 μm, 1 μm, 0.5μm, or less. The conductive particle can have a maximum dimension of atmost about 500 nanometer (nm), 400 nm, 300 nm, 200 nm, 100 nm, 50 nm, 40nm, 30 nm, 20 nm, 10 nm, 9 nm, 8 nm, 7 nm, 6 nm, 5 nm, 4 nm, 3 nm, 2 nm,1 nm, or less. Alternatively, a conductive particle can have a maximumdimension that is greater than about 20 μm. A conductive particle canhave a substantially spherical shape. Alternatively, a conductiveparticle can have a different shape. The conductive particles may beresponsive when exposed to electric fields. The conductive (e.g., metal)content on each conductive particle can be adjusted (e.g., to havehigher or lower percentage) to be more responsive or less responsive tothe same electric field. In some cases, the field-attractable particles115 can comprise both paramagnetic and conductive particles.

The first aqueous fluid 112 can have a substantially uniformconcentration of field-attractable particles 115 as the first aqueousfluid 112 is introduced into junction 110. For example, theconcentration of the field-attractable particles 115 in the firstaqueous fluid 112 in the channel segment 102 at time x can besubstantially uniform with the concentration of field-attractableparticles 115 in the first aqueous fluid 112 in the channel segment 102at time x+δ (where x and δ are positive). In some instances, theconcentration of field-attractable particles 115 can be substantiallyuniform in only the volume of the first aqueous fluid 112, that is, thevolume not including the volume of each of the biological particles 114suspended in the first aqueous fluid 112.

This second fluid 116 can comprise an oil, such as a fluorinated oil,that includes a fluorosurfactant for stabilizing the resulting droplets,e.g., inhibiting subsequent coalescence of the resulting droplets.Examples of particularly useful partitioning fluids andfluorosurfactants are described for example, in U.S. Patent ApplicationPublication No. 2010/0105112, which is entirely incorporated herein byreference for all purposes.

The generated droplets may comprise two subsets of droplets: (1)occupied droplets 118, containing one or more biological particles 114,and (2) unoccupied droplets 120, not containing any biological particles114. Each droplet generated, occupied or unoccupied, may contain somenumber and/or concentration of field-attractable particles 115. In someinstances, the concentration of the field-attractable particles 115 ineach of the unoccupied droplets 120 can be substantially uniform,wherein the concentration is a number of field-attractable particles pertotal droplet volume (and not just the volume of the first aqueous fluid112 in the droplet). In some instances, the concentration of thefield-attractable particles 115 in each of the occupied droplets 118 canbe substantially uniform, wherein the concentration is a number offield-attractable particles per total droplet volume. In otherinstances, as can be easily appreciated, the concentration of thefield-attractable particles 115 in each of the occupied droplets 118 canvary with size and/or the number of biological particles 114 containedin the droplet. In any case, the concentration of field-attractableparticles in any of the unoccupied droplets 120 can be greater than theconcentration of field-attractable particles in any of the occupieddroplets 118 to account for the volume occupied by the biologicalparticle 114 in the occupied droplets 118. In most cases, theconcentration of field-attractable particles in a droplet from theunoccupied droplets 120 can be greater than the concentration offield-attractable particles in a droplet from the occupied droplets 118to account for the volume occupied by the biological particle 114 in theoccupied droplets 118.

For example, assuming that (i) the droplet is spherical and has theradius R_(D), (ii) a biological particle is spherical and has the radiusR₊, and (iii) the concentration of field-attractable particles in thevolume of aqueous fluid is substantially uniform, the ratio of a numberof field-attractable particles in a singularly occupied droplet (N₊) toa number of field-attractable particles in an unoccupied droplet (N)will be:

$\frac{N_{+}}{N_{-}} = {1 - \left( \frac{R_{+}}{R_{D}} \right)^{3}}$

As can be appreciated, the above ratio may change with deviations fromthe above assumptions. For example, an occupied droplet containing threebiological particles can have a ratio of about:

$1 - {3{\left( \frac{R_{+}}{R_{D}} \right)^{3}.}}$

In another aspect, in addition to or as an alternative to droplet basedpartitioning, biological particles may be encapsulated within amicrocapsule, such as a cell bead, that comprises an outer shell orlayer or porous matrix in which is entrained one or more individualbiological particles or small groups of biological particles, and mayinclude other reagents. Encapsulation of biological particles may beperformed by a variety of processes. Such processes combine an aqueousfluid containing the biological particles and also containing thefield-attractable particles to be analyzed with a polymeric precursormaterial that may be capable of being formed into a gel or other solidor semi-solid matrix upon application of a particular stimulus to thepolymer precursor. Such stimuli include, e.g., thermal stimuli (eitherheating or cooling), photo-stimuli (e.g., through photo-curing),chemical stimuli (e.g., through crosslinking, polymerization initiationof the precursor (e.g., through added initiators), or the like.

Preparation of microcapsules comprising biological particles may beperformed by a variety of methods. For example, air knife droplet oraerosol generators may be used to dispense droplets of precursor fluidsinto gelling solutions in order to form microcapsules that includeindividual biological particles or small groups of biological particles.Likewise, membrane based encapsulation systems may be used to generatemicrocapsules comprising encapsulated biological particles as describedherein. Microfluidic systems of the present disclosure, such as thatshown in FIG. 1, may be readily used in encapsulating cells as describedherein, such as to generate a plurality of particles, each particlecomprising field-attractable particles. The plurality of particles maycomprise a first subset of particles occupied by biological particles(e.g., cell beads) and a second subset of particles unoccupied bybiological particles. In particular, and with reference to FIG. 1, theaqueous fluid comprising (i) the biological particles 114, (ii) thefield-attractable particles 115, and (ii) the polymer precursor material(not shown) is flowed into channel junction 110, where it is partitionedinto droplets 118 or 120 comprising or not comprising the individualbiological particles 114, respectively, but always comprising thefield-attractable particles 115, through the flow of non-aqueous fluid116. In the case of encapsulation methods, non-aqueous fluid 116 mayalso include an initiator to cause polymerization and/or crosslinking ofthe polymer precursor to form the microcapsule that includes theentrained biological particles. Examples of polymer precursor/initiatorpairs include those described in U.S. Patent Application Publication No.2014/0378345, which is entirely incorporated herein by reference for allpurposes.

For example, in the case where the polymer precursor material comprisesa linear polymer material, e.g., a linear polyacrylamide, PEG, or otherlinear polymeric material, the activation agent may comprise across-linking agent, or a chemical that activates a cross-linking agentwithin the formed droplets. Likewise, for polymer precursors thatcomprise polymerizable monomers, the activation agent may comprise apolymerization initiator. For example, in certain cases, where thepolymer precursor comprises a mixture of acrylamide monomer with aN,N′-bis-(acryloyl)cystamine (BAC) comonomer, an agent such astetraethylmethylenediamine (TEMED) may be provided within the secondfluid streams in channel segments 104 and 106, which initiates thecopolymerization of the acrylamide and BAC into a cross-linked polymernetwork or, hydrogel.

Upon contact of the second fluid stream 116 with the first fluid stream112 at junction 110 in the formation of droplets, the TEMED may diffusefrom the second fluid 116 into the aqueous first fluid 112 comprisingthe linear polyacrylamide, which will activate the crosslinking of thepolyacrylamide within the droplets, resulting in the formation of thegel, e.g., hydrogel, microcapsules (e.g., droplet 118, 120), as solid orsemi-solid beads or particles entraining the cells 114. Althoughdescribed in terms of polyacrylamide encapsulation, other ‘activatable’encapsulation compositions may also be employed in the context of themethods and compositions described herein. For example, formation ofalginate droplets followed by exposure to divalent metal ions, e.g.,Ca²⁺, can be used as an encapsulation process using the describedprocesses. Likewise, agarose droplets may also be transformed intocapsules through temperature based gelling, e.g., upon cooling, or thelike. In some cases, encapsulated biological particles can beselectively releasable from the microcapsule, e.g., through passage oftime, or upon application of a particular stimulus, that degrades themicrocapsule sufficiently to allow the cell, or its contents to bereleased from the microcapsule, e.g., into a partition, such as adroplet. For example, in the case of the polyacrylamide polymerdescribed above, degradation of the microcapsule may be accomplishedthrough the introduction of an appropriate reducing agent, such as DTTor the like, to cleave disulfide bonds that cross link the polymermatrix (See, e.g., U.S. Patent Application Publication No. 2014/0378345,which is entirely incorporated herein by reference for all purposes).

Encapsulated biological particles (e.g., cell beads) can provide certainpotential advantages of being storable, and more portable than dropletbased partitioned biological particles. Furthermore, in some cases, itmay be desirable to allow biological particles to be analyzed toincubate for a select period of time, in order to characterize changesin such biological particles over time, either in the presence orabsence of different stimuli. In such cases, encapsulation of individualbiological particles may allow for longer incubation than partitioningin emulsion droplets, although in some cases, droplet partitionedbiological particles may also be incubated for different periods oftime, e.g., at least 10 seconds, at least 30 seconds, at least 1 minute,at least 5 minutes, at least 10 minutes, at least 30 minutes, at least 1hour, at least 2 hours, at least 5 hours, or at least 10 hours or more.The encapsulation of biological particles may constitute thepartitioning of the biological particles into which other reagents areco-partitioned. Alternatively, encapsulated biological particles may bereadily deposited into other partitions, e.g., droplets, as describedabove.

In accordance with certain aspects, the biological particles may bepartitioned along with lysis reagents in order to release the contentsof the biological particles within the partition. In such cases, thelysis agents can be contacted with the biological particle suspensionconcurrently with, or immediately prior to the introduction of thebiological particles into the partitioning junction/droplet generationzone, e.g., through an additional channel or channels upstream ofchannel junction 110. Examples of lysis agents include bioactivereagents, such as lysis enzymes that are used for lysis of differentcell types, e.g., gram positive or negative bacteria, plants, yeast,mammalian, etc., such as lysozymes, achromopeptidase, lysostaphin,labiase, kitalase, lyticase, and a variety of other lysis enzymesavailable from, e.g., Sigma-Aldrich, Inc. (St Louis, Mo.), as well asother commercially available lysis enzymes. Other lysis agents mayadditionally or alternatively be co-partitioned with the biologicalparticles to cause the release of the biological particles's contentsinto the partitions. For example, in some cases, surfactant based lysissolutions may be used to lyse cells, although these may be lessdesirable for emulsion based systems where the surfactants can interferewith stable emulsions. In some cases, lysis solutions may includenon-ionic surfactants such as, for example, TritonX-100 and Tween 20. Insome cases, lysis solutions may include ionic surfactants such as, forexample, sarcosyl and sodium dodecyl sulfate (SDS). Electroporation,thermal, acoustic or mechanical cellular disruption may also be used incertain cases, e.g., non-emulsion based partitioning such asencapsulation of biological particles that may be in addition to or inplace of droplet partitioning, where any pore size of the encapsulate issufficiently small to retain nucleic acid fragments of a desired size,following cellular disruption.

In addition to the lysis agents co-partitioned with the biologicalparticles described above, other reagents can also be co-partitionedwith the biological particles, including, for example, DNase and RNaseinactivating agents or inhibitors, such as proteinase K, chelatingagents, such as EDTA, and other reagents employed in removing orotherwise reducing negative activity or impact of different cell lysatecomponents on subsequent processing of nucleic acids. In addition, inthe case of encapsulated biological particles, the biological particlesmay be exposed to an appropriate stimulus to release the biologicalparticles or their contents from a co-partitioned microcapsule. Forexample, in some cases, a chemical stimulus may be co-partitioned alongwith an encapsulated biological particle to allow for the degradation ofthe microcapsule and release of the cell or its contents into the largerpartition. In some cases, this stimulus may be the same as the stimulusdescribed elsewhere herein for release of oligonucleotides from theirrespective microcapsule (e.g., bead). In alternative aspects, this maybe a different and non-overlapping stimulus, in order to allow anencapsulated biological particle to be released into a partition at adifferent time from the release of oligonucleotides into the samepartition.

Additional reagents may also be co-partitioned with the biologicalparticles, such as endonucleases to fragment a biological particle'sDNA, DNA polymerase enzymes and dNTPs used to amplify the biologicalparticle's nucleic acid fragments and to attach the barcode moleculartags to the amplified fragments. Additional reagents may also includereverse transcriptase enzymes, including enzymes with terminaltransferase activity, primers and oligonucleotides, and switcholigonucleotides (also referred to herein as “switch oligos” or“template switching oligonucleotides”) which can be used for templateswitching. In some cases, template switching can be used to increase thelength of a cDNA. In some cases, template switching can be used toappend a predefined nucleic acid sequence to the cDNA. In an example oftemplate switching, cDNA can be generated from reverse transcription ofa template, e.g., cellular mRNA, where a reverse transcriptase withterminal transferase activity can add additional nucleotides, e.g.,polyC, to the cDNA in a template independent manner. Switch oligos caninclude sequences complementary to the additional nucleotides, e.g.,polyG. The additional nucleotides (e.g., polyC) on the cDNA canhybridize to the additional nucleotides (e.g., polyG) on the switcholigo, whereby the switch oligo can be used by the reverse transcriptaseas template to further extend the cDNA. Template switchingoligonucleotides may comprise a hybridization region and a templateregion. The hybridization region can comprise any sequence capable ofhybridizing to the target. In some cases, as previously described, thehybridization region comprises a series of G bases to complement theoverhanging C bases at the 3′ end of a cDNA molecule. The series of Gbases may comprise 1 G base, 2 G bases, 3 G bases, 4 G bases, 5 G basesor more than 5 G bases. The template sequence can comprise any sequenceto be incorporated into the cDNA. In some cases, the template regioncomprises at least 1 (e.g., at least 2, 3, 4, 5 or more) tag sequencesand/or functional sequences. Switch oligos may comprise deoxyribonucleicacids; ribonucleic acids; modified nucleic acids including2-Aminopurine, 2,6-Diaminopurine (2-Amino-dA), inverted dT, 5-Methyl dC,2′-deoxyInosine, Super T (5-hydroxybutynl-2′-deoxyuridine), Super G(8-aza-7-deazaguanosine), locked nucleic acids (LNAs), unlocked nucleicacids (UNAs, e.g., UNA-A, UNA-U, UNA-C, UNA-G), Iso-dG, Iso-dC, 2′Fluoro bases (e.g., Fluoro C, Fluoro U, Fluoro A, and Fluoro G), or anycombination.

In some cases, the length of a switch oligo may be 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44,45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62,63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80,81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98,99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112,113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126,127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140,141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154,155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168,169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182,183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196,197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210,211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224,225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238,239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250 nucleotidesor longer.

In some cases, the length of a switch oligo may be at least 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42,43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60,61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78,79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96,97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111,112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125,126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139,140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153,154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167,168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181,182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195,196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209,210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223,224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237,238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249 or 250nucleotides or longer.

In some cases, the length of a switch oligo may be at most 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42,43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60,61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78,79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96,97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111,112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125,126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139,140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153,154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167,168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181,182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195,196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209,210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223,224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237,238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249 or 250nucleotides.

Once the contents of the cells are released into their respectivepartitions, the macromolecular components (e.g., macromolecularconstituents of biological particles, such as RNA, DNA, or proteins)contained therein may be further processed within the partitions. Inaccordance with the methods and systems described herein, themacromolecular component contents of individual biological particles canbe provided with unique identifiers such that, upon characterization ofthose macromolecular components they may be attributed as having beenderived from the same biological particle or particles. The ability toattribute characteristics to individual biological particles or groupsof biological particles is provided by the assignment of uniqueidentifiers specifically to an individual biological particle or groupsof biological particles. Unique identifiers, e.g., in the form ofnucleic acid barcodes, can be assigned or associated with individualbiological particles or populations of biological particle, in order totag or label the biological particle's macromolecular components (and asa result, its characteristics) with the unique identifiers. These uniqueidentifiers can then be used to attribute the biological particle'scomponents and characteristics to an individual biological particle orgroup of biological particles. In some aspects, this is performed byco-partitioning the individual biological particle or groups ofbiological particles with the unique identifiers. In some aspects, theunique identifiers are provided in the form of oligonucleotides thatcomprise nucleic acid barcode sequences that may be attached to orotherwise associated with the nucleic acid contents of individualbiological particle, or to other components of the biological particle,and particularly to fragments of those nucleic acids. Theoligonucleotides are partitioned such that as between oligonucleotidesin a given partition, the nucleic acid barcode sequences containedtherein are the same, but as between different partitions, theoligonucleotides can, and do, have differing barcode sequences, or atleast represent a large number of different barcode sequences across allof the partitions in a given analysis. In some aspects, only one nucleicacid barcode sequence can be associated with a given partition, althoughin some cases, two or more different barcode sequences may be present.

The nucleic acid barcode sequences can include from 6 to about 20 ormore nucleotides within the sequence of the oligonucleotides. In somecases, the length of a barcode sequence may be 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20 nucleotides or longer. In some cases, thelength of a barcode sequence may be at least 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20 nucleotides or longer. In some cases, thelength of a barcode sequence may be at most 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20 nucleotides or shorter. These nucleotides maybe completely contiguous, i.e., in a single stretch of adjacentnucleotides, or they may be separated into two or more separatesubsequences that are separated by 1 or more nucleotides. In some cases,separated barcode subsequences can be from about 4 to about 16nucleotides in length. In some cases, the barcode subsequence may be 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 nucleotides or longer. In somecases, the barcode subsequence may be at least 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16 nucleotides or longer. In some cases, the barcodesubsequence may be at most 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16nucleotides or shorter.

The co-partitioned oligonucleotides can also comprise other functionalsequences useful in the processing of the nucleic acids from theco-partitioned biological particles. These sequences include, e.g.,targeted or random/universal amplification primer sequences foramplifying the genomic DNA from the individual biological particleswithin the partitions while attaching the associated barcode sequences,sequencing primers or primer recognition sites, hybridization or probingsequences, e.g., for identification of presence of the sequences or forpulling down barcoded nucleic acids, or any of a number of otherpotential functional sequences. Other mechanisms of co-partitioningoligonucleotides may also be employed, including, e.g., coalescence oftwo or more droplets, where one droplet contains oligonucleotides, ormicrodispensing of oligonucleotides into partitions, e.g., dropletswithin microfluidic systems.

In an example, microcapsules, such as beads (e.g., see FIG. 19), areprovided that each includes large numbers of the above describedbarcoded oligonucleotides releasably attached to the beads, where all ofthe oligonucleotides attached to a particular bead will include the samenucleic acid barcode sequence, but where a large number of diversebarcode sequences are represented across the population of beads used.In some embodiments, hydrogel beads, e.g., comprising polyacrylamidepolymer matrices, are used as a solid support and delivery vehicle forthe oligonucleotides into the partitions, as they are capable ofcarrying large numbers of oligonucleotide molecules, and may beconfigured to release those oligonucleotides upon exposure to aparticular stimulus, as described elsewhere herein. In some cases, thepopulation of beads will provide a diverse barcode sequence library thatincludes at least about 1,000 different barcode sequences, at leastabout 5,000 different barcode sequences, at least about 10,000 differentbarcode sequences, at least about 50,000 different barcode sequences, atleast about 100,000 different barcode sequences, at least about1,000,000 different barcode sequences, at least about 5,000,000different barcode sequences, or at least about 10,000,000 differentbarcode sequences, or more. Additionally, each bead can be provided withlarge numbers of oligonucleotide molecules attached. In particular, thenumber of molecules of oligonucleotides including the barcode sequenceon an individual bead can be at least about 1,000 oligonucleotidemolecules, at least about 5,000 oligonucleotide molecules, at leastabout 10,000 oligonucleotide molecules, at least about 50,000oligonucleotide molecules, at least about 100,000 oligonucleotidemolecules, at least about 500,000 oligonucleotides, at least about1,000,000 oligonucleotide molecules, at least about 5,000,000oligonucleotide molecules, at least about 10,000,000 oligonucleotidemolecules, at least about 50,000,000 oligonucleotide molecules, at leastabout 100,000,000 oligonucleotide molecules, and in some cases at leastabout 1 billion oligonucleotide molecules, or more.

Moreover, when the population of beads is partitioned, the resultingpopulation of partitions can also include a diverse barcode library thatincludes at least about 1,000 different barcode sequences, at leastabout 5,000 different barcode sequences, at least about 10,000 differentbarcode sequences, at least at least about 50,000 different barcodesequences, at least about 100,000 different barcode sequences, at leastabout 1,000,000 different barcode sequences, at least about 5,000,000different barcode sequences, or at least about 10,000,000 differentbarcode sequences. Additionally, each partition of the population caninclude at least about 1,000 oligonucleotide molecules, at least about5,000 oligonucleotide molecules, at least about 10,000 oligonucleotidemolecules, at least about 50,000 oligonucleotide molecules, at leastabout 100,000 oligonucleotide molecules, at least about 500,000oligonucleotides, at least about 1,000,000 oligonucleotide molecules, atleast about 5,000,000 oligonucleotide molecules, at least about10,000,000 oligonucleotide molecules, at least about 50,000,000oligonucleotide molecules, at least about 100,000,000 oligonucleotidemolecules, and in some cases at least about 1 billion oligonucleotidemolecules.

In some cases, it may be desirable to incorporate multiple differentbarcodes within a given partition, either attached to a single ormultiple beads within the partition. For example, in some cases, amixed, but known barcode sequences set may provide greater assurance ofidentification in the subsequent processing, e.g., by providing astronger address or attribution of the barcodes to a given partition, asa duplicate or independent confirmation of the output from a givenpartition.

The oligonucleotides are releasable from the beads upon the applicationof a particular stimulus to the beads. In some cases, the stimulus maybe a photo-stimulus, e.g., through cleavage of a photo-labile linkagethat releases the oligonucleotides. In other cases, a thermal stimulusmay be used, where elevation of the temperature of the beads environmentwill result in cleavage of a linkage or other release of theoligonucleotides form the beads. In still other cases, a chemicalstimulus is used that cleaves a linkage of the oligonucleotides to thebeads, or otherwise results in release of the oligonucleotides from thebeads. In one case, such compositions include the polyacrylamidematrices described above for encapsulation of biological particles, andmay be degraded for release of the attached oligonucleotides throughexposure to a reducing agent, such as DTT.

For example, in FIG. 1, concurrent to the stream of the first aqueousfluid 112 flowing through channel 102 towards the junction 110, a secondaqueous stream comprising barcode carrying beads suspended in a thirdfluid can be flowed through another channel (not shown in FIG. 1)towards the junction 110. The third fluid can be the same fluid materialas the first aqueous fluid 112. A non-aqueous partitioning fluid 116 isintroduced into channel junction 110 from each of side channels 104 and106, and the combined streams are flowed into outlet channel 108. Withinchannel junction 110, the two combined aqueous streams from channelsegments 102 and the other channel carrying the third fluid arecombined, and partitioned into droplets 118, 120. The occupied dropletsmay contain either one or more biological particles, either one or morebarcode carrying beads, or both at least a biological particle and atleast a barcode carrying bead. The unoccupied droplets may containneither biological particles nor barcode carrying beads. However, alldroplets, both occupied and unoccupied droplets, can comprise at leastsome concentration of field-attractable particles 115. As notedpreviously, by controlling the flow characteristics of each of thefluids combining at channel junction 110, as well as controlling thegeometry of the channel junction, partitioning can be optimized toachieve a desired occupancy level of beads, biological particles, orboth, within the partitions that are generated.

In some cases, assuming that (i) the droplet is spherical and has theradius R_(D), (ii) a biological particle is spherical and has the radiusR₊, (iii) a barcode carrying bead is spherical and has the radius R_(B)and (iv) the concentration of field-attractable particles in the volumeof aqueous fluid is substantially uniform, the ratio of a number offield-attractable particles in a singularly occupied droplet (N_(+,B))(containing one of each of a biological particle and a barcode carryingbead) to a number of field-attractable particles in an unoccupieddroplet (N⁻) will be:

$\frac{N_{+ {,B}}}{N_{-}} = {1 - \left( \frac{R_{+}}{R_{D}} \right)^{3} - \left( \frac{R_{B}}{R_{D}} \right)^{3}}$

FIG. 2A shows an example of a microfluidic channel structure forseparating occupied droplets from unoccupied droplets. As describedelsewhere herein, when droplets are generated, there may be a firstsubset population of occupied droplets containing one or more biologicalparticles and a second subset population of unoccupied droplets notcontaining any biological particles. In some cases, the droplets mayadditionally contain one or more barcode carrying beads. For example, adroplet may have only a biological particle, a droplet may have only abarcode carrying bead, a droplet may have both a biological particle anda barcode carrying bead, or a droplet may have neither biologicalparticles nor barcode carrying beads. In some cases, the majority ofoccupied partitions can include no more than one biological particle peroccupied partition and, in some cases, some of the generated partitionscan be unoccupied (of any biological particle). In some cases, though,some of the occupied partitions may include more than one biologicalparticle. In some cases, the partitioning process may be controlled suchthat fewer than 25% of the occupied partitions contain more than onebiological particle, and in many cases, fewer than 20% of the occupiedpartitions have more than one biological particle, while in some cases,fewer than 10% or even fewer than 5% of the occupied partitions includemore than one biological particle per partition.

As shown in FIG. 2A, the channel structure can include channel segments202, 204, and 206 meeting at a channel intersection 211. In someinstances, the outflow channel 108 of the emulsion carrying thegenerated droplets in FIG. 1 can be upstream of the channel segment 202,such that the generated droplets are directed to flow to the channelintersection 211 for subsequent sorting. A controller 220 can beoperatively coupled to a fluid flow unit 218, to facilitate flow offluid in the channel structure, and a field application unit 216, toapply one or more fields to the channel structure.

In operation, a plurality of discrete droplets, each comprising a firstaqueous fluid 210 can flow as emulsions in a second fluid 208, whereinthe second fluid 208 is immiscible to the first aqueous fluid 210. Thedroplets being transported along channel segment 202 into intersection211 can comprise a first subset of droplets 214 that are each occupiedwith at least a biological particle and/or a barcode carrying bead and asecond subset of droplets 212 that are each unoccupied. Every droplet,including occupied and unoccupied droplets, can comprise someconcentration of field-attractable particles. As described above, agiven unoccupied droplet can have a higher concentration offield-attractable particles than a given occupied droplet to account forthe volume occupied by a biological particle and/or a barcode bead in anoccupied droplet.

After sorting at or near the intersection 211, the first subset ofdroplets 214 can be directed to flow along channel segment 206 and awayfrom the intersection 211, and the second subset of droplets 212 can bedirected to flow along channel segment 204 and away from theintersection 211.

The fluid flow unit 218 can be configured to subject the second fluid208 containing a plurality of droplets, including both occupied dropletsand unoccupied droplets, to flow along the channel 202 towards theintersection 211. The fluid flow unit 218 can be configured to subjectthe second fluid 208 containing a plurality of droplets, wherein amajority of the droplets is unoccupied droplets, to flow along thechannel 204 away from the intersection 211. The fluid flow unit 218 canbe configured to subject the second fluid 208 containing a plurality ofdroplets, wherein a majority of the droplets is occupied droplets, toflow along the channel 206 away from the intersection 211.Alternatively, the fluid flow unit 218 can be configured to subject thesecond fluid 208 containing a plurality of droplets, wherein a majorityof the droplets is unoccupied droplets, to flow along the channel 206away from the intersection 211, and configured to subject the secondfluid 208 containing a plurality of droplets, wherein a majority of thedroplets is occupied droplets, to flow along the channel 204 away fromthe intersection 211. The fluid flow unit 218 can be operatively coupledto the controller 220. For example, the fluid flow unit 218 may receiveinstructions from the controller 220 regarding fluid pressure and/orvelocity.

In some instances, the fluid flow unit 218 may comprise a compressor toprovide positive pressure at an upstream location to direct the fluidfrom the upstream location to flow to a downstream location. In someinstances, the fluid flow unit 218 may comprise a pump to providenegative pressure at a downstream location to direct the fluid from anupstream location to flow to the downstream location. In some instances,the fluid flow unit 218 may comprise both a compressor and a pump, eachat different locations. In some instances, the fluid flow unit 218 maycomprise different devices at different locations. The fluid flow unit218 may comprise an actuator. While FIG. 2A depicts one fluid flow unit218, it may be appreciated that there may be a plurality of fluid flowunits 218, each in communication with the controller 220 and/or witheach other. For example, there can be a separate fluid flow unit todirect the fluid in channel 202 towards the intersection 211, a separatefluid flow unit to direct the fluid in channel 204 away from theintersection 211, and a separate fluid flow unit to direct the fluid inchannel 206 away from the intersection 211.

The field application unit 216 can be configured to apply a force fieldto the channel structure. In some instances, the field application unit216 can be configured to apply a force field at or near the intersection211 such that the second subset of droplets (unoccupied droplets) aregenerally directed along the channel segment 204 and away from theintersection 211, and the first subset of droplets (occupied droplets)are generally directed along the channel segment 206 and away from theintersection 211, thereby isolating the two subsets of droplets.

For example, the field application unit 216 can apply a magnetic fieldat or near the intersection 211. The field application unit 216 can be amagnet and/or a circuit (e.g., current carrying device) configured togenerate a magnetic field. On account of each droplet containingfield-attractable particles (e.g., paramagnetic particles), each dropletmay be attracted (e.g., due to paramagnetic particles) or repelled(e.g., due to diamagnetic particles) to or away, respectively, from themagnetic field. The degree of attraction (or repulsion) can beproportional to a number (and/or a concentration) of field-attractableparticles in each droplet. That is, the magnetic force acting on adroplet, from the same magnetic field, can be proportional to a number(and/or a concentration) of field-attractable particles in the droplet.As previously described above, assuming that (i) the droplet isspherical and has the radius R_(D), (ii) a biological particle isspherical and has the radius R₊, and (iii) the concentration offield-attractable particles in the volume of aqueous fluid issubstantially uniform, the ratio of a number of field-attractableparticles in a singularly occupied droplet (N₊) (wherein the occupieddroplet contains a single biological particle) to a number offield-attractable particles in an unoccupied droplet (N⁻) will be, andthus the ratio of a magnetic force acting on a singularly occupieddroplet (F_(M+)) to a magnetic force acting on an unoccupied droplet(F_(M−)) will be:

$\frac{N_{+}}{N_{-}} = {\frac{F_{M +}}{F_{M -}} = {1 - \left( \frac{R_{+}}{R_{D}} \right)^{3}}}$

That is, there may be a stronger (differential) force acting on a givenunoccupied droplet than a given occupied droplet. As can be appreciated,the above ratio may change with deviations from the above assumptions(e.g., non-spherical biological particle, non-spherical droplet,non-uniform concentration of field-attractable particles in volume ofaqueous fluid, etc.).

In another example, the field application unit 216 can apply an electricfield at or near the intersection 211. On account of each dropletcontaining field-attractable particles (e.g., conductive particles),each droplet may be attracted or repelled to or away, respectively, fromthe electric field. The degree of attraction (or repulsion) can beproportional to a number (and/or a concentration) of field-attractableparticles in each droplet. That is, the electric force acting on adroplet, from the same electric field, can be proportional to a number(and/or a concentration) of field-attractable particles in the droplet.As previously described above, assuming that (i) the droplet isspherical and has the radius R_(D), (ii) a biological particle isspherical and has the radius R₊, and (iii) the concentration offield-attractable particles in the volume of aqueous fluid issubstantially uniform, the ratio of a number of field-attractableparticles in a singularly occupied droplet (N₊) (wherein the occupieddroplet contains a single biological particle) to a number offield-attractable particles in an unoccupied droplet (N⁻) will be, andthus the ratio of an electric force acting on a singularly occupieddroplet (F_(E+)) to an electric force acting on an unoccupied droplet(F_(E)) will be:

$\frac{N_{+}}{N_{-}} = {\frac{F_{E +}}{F_{E -}} = {1 - \left( \frac{R_{+}}{R_{D}} \right)^{3}}}$

As can be appreciated, the above ratio may change with deviations fromthe above assumptions (e.g., non-spherical biological particle,non-spherical droplet, non-uniform concentration of field-attractableparticles in volume of aqueous fluid, etc.). In some instances, thefluid flow unit 218 can apply both an electric field and a magneticfield.

The field application unit 216 can be operatively coupled to thecontroller 220. For example, the field application unit 216 may receiveinstructions from the controller 220 regarding force field strength,orientation, frequency, and/or other variables. While FIG. 2A depictsone field application unit 216, it may be appreciated that there may bea plurality of field application units 216, each in communication withthe controller 220, other controllers, and/or with each other. Forexample, there can be a plurality of field application units, eachlocated at a different location. The controller 220 may instruct thefield application unit 216 to apply a force field sufficiently strongand in a sufficiently targeted direction towards the mixed (occupied andunoccupied) droplets such as to direct the unoccupied droplets in onechannel and direct the occupied droplets to another channel. In anexample, the field application unit 216 can be placed in a locationcloser to a first channel (e.g., channel 204) than a second channel(e.g., channel 206) to direct the unoccupied droplets (which are subjectto a stronger force from the same field) to the first channel, assumingthat the field is strongest when closest to the field application unit216. The stronger a force from the field acts on a droplet, the morelikely that the droplet will deviate from an initial flow direction(e.g., direction of flow in channel 202) into another channel havinganother direction. In some instances, the field application unit may belocated at least in part downstream, from the intersection 211, of achannel intended to isolate unoccupied droplets (e.g., channel 204).

For example, a force field applied can be strong enough to direct theunoccupied droplets to flow to a first channel but weak enough to direct(or leave be) the occupied droplets to flow to a second channel. In someinstances, a magnetic field applied by the field application unit 216can have a magnetic flux density range from at least about 10⁻⁵ Teslas(T) to about 1 T. Alternatively, the magnetic flux density can be lessthan or equal to about 10⁻⁵ T and/or greater than or equal to about 1 T.In some instances, an electric field applied by the field applicationunit 216 can have an electric field strength of at least about 1 voltper meter (V/m), 2 V/m, 3 V/m, 4 V/m, 5 V/m, 10 V/m, or more.Alternatively, the electric field strength can be less than about 10V/m, 5 V/m, 4 V/m, 3 V/m, 2 V/m, 1 V/m, or less.

FIG. 2B shows an example of a multi-stage microfluidic channel structurefor separating singularly occupied droplets. As described elsewhereherein, when droplets are generated, there may be a first subsetpopulation of occupied droplets containing a single biological particle(e.g., cell), a second subset population of unoccupied droplets notcontaining any biological particles, and a third subset population ofoccupied droplets containing multiple (e.g., two or more) biologicalparticles. In some cases, the droplets may additionally contain one ormore barcode carrying beads. For example, a droplet may have onlybiological particle(s), a droplet may have only barcode carryingbead(s), a droplet may have both biological particle(s) and barcodecarrying bead(s), or a droplet may have neither biological particle(s)nor barcode carrying bead(s). The generated droplets may be sorted toisolate the first subset population, second subset population, and thirdsubset population in multiple stages.

As shown in FIG. 2B, the channel structure can include channel segments222, 224, 226, 244, and 246. Channel segments 222, 224, and 226 may meetat channel intersection 231. Channel segments 226, 244, and 246 may meetat channel intersection 233. In some instances, the outflow channel 108of the emulsion carrying the generated droplets in FIG. 1 can beupstream of the channel segment 222, such that the generated dropletsare directed to flow to the channel intersection 231 for subsequentsorting. The generated droplets may comprise a first subset populationof singularly occupied droplets (e.g., 230A), a second subset populationof unoccupied droplets (e.g., 230B), and a third subset population ofmultiply occupied droplets (e.g., 230C). A first controller 240 can beoperatively coupled to a fluid flow unit 238, to facilitate flow offluid in the channel structure, and a first field application unit 236,to apply one or more fields to the channel structure. A secondcontroller 241 can be operatively coupled to a second field applicationunit 237, to apply one or more fields to the channel structure.

In operation, a plurality of discrete droplets can flow as emulsions ina fluid 228. The droplets being transported along channel segment 222into intersection 231 can comprise the first subset population ofsingularly occupied droplets (e.g., 230A), the second subset populationof unoccupied droplets (e.g., 230B), and the third subset population ofmultiply occupied droplets (e.g., 230C). Every droplet, includingsingularly occupied, multiply occupied, and unoccupied droplets, cancomprise some concentration of field-attractable particles. As describedabove, a given unoccupied droplet can have a higher concentration offield-attractable particles than a given occupied droplet to account forthe volume occupied by a biological particle and/or a barcode bead in anoccupied droplet. Within occupied droplets, a given singly occupieddroplet can have a higher concentration of field-attractable particlesthan a given multiply occupied droplet to account for the differentialvolume occupied by the different numbers of biological particles (and/orbarcode beads) in the droplet.

After a first stage of sorting at or near the intersection 231, thefirst and third subsets of droplets (e.g., 230A, C) can be directed toflow along channel segment 226 and away from the intersection 231, andthe second subset of droplets 230B can be directed to flow along channelsegment 224 and away from the intersection 231.

The fluid flow unit 238 can be configured to subject the fluid 228containing a plurality of droplets including the occupied and/orunoccupied droplets (e.g., as emulsion suspensions) to or fromintersections 231, 233 along channel segments 222, 224, 226, 244, and246. The fluid flow unit 238 can be operatively coupled to the firstcontroller 240. For example, the fluid flow unit 238 may receiveinstructions from the first controller 240 regarding fluid pressureand/or velocity.

In some instances, the fluid flow unit 238 may comprise a compressor toprovide positive pressure at an upstream location to direct the fluidfrom the upstream location to flow to a downstream location. In someinstances, the fluid flow unit 238 may comprise a pump to providenegative pressure at a downstream location to direct the fluid from anupstream location to flow to the downstream location. In some instances,the fluid flow unit 238 may comprise both a compressor and a pump, eachat different locations. In some instances, the fluid flow unit 238 maycomprise different devices at different locations. The fluid flow unit238 may comprise an actuator. While FIG. 2B depicts one fluid flow unit238, it may be appreciated that there may be a plurality of fluid flowunits 238, each in communication with the first controller 240, othercontrollers (e.g., second controller 241), and/or with each other. Forexample, there can be a separate fluid flow unit to direct the fluid inchannel 222 towards the intersection 231, a separate fluid flow unit todirect the fluid in channel 224 away from the intersection 231, and aseparate fluid flow unit to direct the fluid in channel 226 away fromthe intersection 231.

The first field application unit 236 can be configured to apply a forcefield to the channel structure. In some instances, the field applicationunit 236 can be configured to apply a force field at or near theintersection 231 such that the second subset of droplets (unoccupieddroplets) are generally directed along the channel segment 224 and awayfrom the intersection 231, and the first and third subset of droplets(occupied droplets) are generally directed along the channel segment 226and away from the intersection 231, thereby separating the subsets ofdroplets. For example, as described elsewhere herein, the first fieldapplication unit 236 may apply a magnetic field at or near theintersection 231. On account of each droplet containingfield-attractable particles (e.g., paramagnetic particles), each dropletmay be attracted (e.g., due to paramagnetic particles) or repelled(e.g., due to diamagnetic particles) to or away, respectively, from themagnetic field. The degree of attraction (or repulsion) can beproportional to a number (and/or a concentration) of field-attractableparticles in each droplet. That is, the magnetic force acting on adroplet, from the same magnetic field, can be proportional to a number(and/or a concentration) of field-attractable particles in the droplet.As described above, assuming that (i) the droplet is spherical and hasthe radius R_(D), (ii) a biological particle is spherical and has theradius R₊, and (iii) the concentration of field-attractable particles inthe volume of aqueous fluid is substantially uniform, the ratio of anumber of field-attractable particles in a singularly occupied droplet(N₊) (wherein the occupied droplet contains a single biologicalparticle) to a number of field-attractable particles in an unoccupieddroplet (N) will be, and thus the ratio of a magnetic force acting on asingularly occupied droplet (F_(M+)) to a magnetic force acting on anunoccupied droplet (F_(M−)) will be:

$\frac{N_{+}}{N_{-}} = {\frac{F_{M +}}{F_{M -}} = {1 - \left( \frac{R_{+}}{R_{D}} \right)^{3}}}$

Similarly, the ratio of a number of field-attractable particles in adoubly occupied droplet (N₂₊) (wherein the occupied droplet contains twobiological particles) to a number of field-attractable particles in anunoccupied droplet (N) will be, and thus the ratio of a magnetic forceacting on a doubly occupied droplet (F_(M,2+)) to a magnetic forceacting on an unoccupied droplet (F_(M−)) will be:

$\frac{N_{2 +}}{N_{-}} = {\frac{F_{M,{2 +}}}{F_{M -}} = {1 - {2\left( \frac{R_{+}}{R_{D}} \right)^{3}}}}$

That is, there may be a stronger (differential) force acting on a givenunoccupied droplet than a given occupied droplet. Between occupieddroplets, there may be a stronger (differential) force acting on a givensingularly occupied droplet than a given doubly occupied droplet.Similarly, the more occupied a droplet is (with more biologicalparticles), the less it will be affected by the magnetic field. As canbe appreciated, the above ratios may change with deviations from theabove assumptions (e.g., non-spherical biological particle,non-spherical droplet, non-uniform concentration of field-attractableparticles in volume of aqueous fluid, etc.).

In another example, as described elsewhere herein, the first fieldapplication unit 236 can apply an electric field at or near theintersection 231. On account of each droplet containingfield-attractable particles (e.g., conductive particles), each dropletmay be attracted or repelled to or away, respectively, from the electricfield. The degree of attraction (or repulsion) can be proportional to anumber (and/or a concentration) of field-attractable particles in eachdroplet. That is, the electric force acting on a droplet, from the sameelectric field, can be proportional to a number (and/or a concentration)of field-attractable particles in the droplet.

The first field application unit 236 can be operatively coupled to thefirst controller 240. For example, the first field application unit 236may receive instructions from the first controller 240 regarding forcefield strength, orientation, frequency, and/or other variables. Thefirst controller 240 may instruct the first field application unit 236to apply a force field sufficiently strong and in a sufficientlytargeted direction towards the mixed (occupied and unoccupied) dropletssuch as to direct the unoccupied droplets in one channel and direct theoccupied droplets to another channel. In an example, the first fieldapplication unit 236 can be placed in a location closer to a firstchannel (e.g., channel 224) than a second channel (e.g., channel 226) todirect the unoccupied droplets (which are subject to a stronger forcefrom the same field) to the first channel, assuming that the field isstrongest when closest to the first field application unit 236. Thestronger a force from the field acts on a droplet, the more likely thatthe droplet will deviate from an initial flow direction (e.g., directionof flow in channel 222) into another channel having another direction.In some instances, the field application unit may be located at least inpart downstream, from the intersection 231, of a channel intended toisolate unoccupied droplets (e.g., channel 224).

For example, a force field applied can be strong enough to direct theunoccupied droplets to flow to a first channel but weak enough to direct(or leave be) the occupied droplets (whether singularly, doubly, orotherwise multiply occupied) to flow to a second channel. In someinstances, a magnetic field applied by the field application unit 236can have a magnetic flux density range from at least about 10⁻⁵ Teslas(T) to about 1 T. Alternatively, the magnetic flux density can be lessthan or equal to about 10⁻⁵ T and/or greater than or equal to about 1 T.In some instances, an electric field applied by the field applicationunit 236 can have an electric field strength of at least about 1 voltper meter (V/m), 2 V/m, 3 V/m, 4 V/m, 5 V/m, 10 V/m, or more.Alternatively, the electric field strength can be less than about 10V/m, 5 V/m, 4 V/m, 3 V/m, 2 V/m, 1 V/m, or less.

The first and third sets of droplets (e.g., 230A, C) isolated to channel226 may be subjected to a second stage of sorting at or near theintersection 233. After the second stage of sorting at or near theintersection 233, the third subset of droplets (e.g., 230C) can bedirected to flow along channel segment 246 and away from theintersection 233, and the first subset of droplets 230A can be directedto flow along channel segment 244 and away from the intersection 233.Thus, the singularly occupied droplets (first subset of droplets) may beisolated from both the unoccupied droplets and the multiply occupieddroplets.

The second field application unit 237 can be configured to apply a forcefield to the channel structure. In some instances, the field applicationunit 237 can be configured to apply a force field at or near theintersection 233 such that the first subset of droplets (singularlyoccupied droplets) are generally directed along the channel segment 244and away from the intersection 233, and the third subset of droplets(multiply occupied droplets) are generally directed along the channelsegment 246 and away from the intersection 233, thereby separating thefirst and third subsets of droplets. For example, as described elsewhereherein, the second field application unit 237 may apply a magnetic fieldat or near the intersection 233. As described elsewhere herein, asbetween occupied droplets, there may be a stronger (differential) forceacting on a given singularly occupied droplet than a given doublyoccupied droplet. Similarly, the more occupied a droplet is (with morebiological particles), the less it will be affected by the magneticfield. In another example, as described elsewhere herein, the secondfield application unit 237 can apply an electric field at or near theintersection 233.

The second field application unit 237 can be operatively coupled to thesecond controller 241. For example, the second field application unit237 may receive instructions from the second controller 241 regardingforce field strength, orientation, frequency, and/or other variables.The second controller 241 may instruct the second field application unit237 to apply a force field sufficiently strong and in a sufficientlytargeted direction towards the mixed (singularly occupied and multiplyoccupied) droplets such as to direct the singularly droplets in onechannel and direct the multiply occupied droplets to another channel. Inan example, the second field application unit 237 can be placed in alocation closer to a first channel (e.g., channel 244) than a secondchannel (e.g., channel 246) to direct the singularly occupied droplets(which are subject to a stronger force from the same field than multiplyoccupied droplets) to the first channel, assuming that the field isstrongest when closest to the second field application unit 237. Thestronger a force from the field acts on a droplet, the more likely thatthe droplet will deviate from an initial flow direction (e.g., directionof flow in channel 226) into another channel having another direction.In some instances, the field application unit may be located at least inpart downstream, from the intersection 233, of a channel intended toisolate singularly occupied droplets (e.g., channel 244). Alternativelyor in addition to, the second field application unit 236 can beoperatively coupled to the first controller 240.

For example, a force field applied can be strong enough to direct thesingularly occupied droplets to flow to a first channel but weak enoughto direct (or leave be) the multiply occupied droplets (whethersingularly, doubly, or otherwise multiply occupied) to flow to a secondchannel. In some instances, the force field applied by the second fieldapplication unit 237 may be stronger than the force field applied by thefirst field application unit 236. In some instances, a magnetic fieldapplied by the second field application unit 237 can have a magneticflux density range from at least about 10⁻⁵ Teslas (T) to about 1 T.Alternatively, the magnetic flux density can be less than or equal toabout 10⁻⁵ T and/or greater than or equal to about 1 T. In someinstances, an electric field applied by the field application unit 236can have an electric field strength of at least about 1 volt per meter(V/m), 2 V/m, 3 V/m, 4 V/m, 5 V/m, 10 V/m, or more. Alternatively, theelectric field strength can be less than about 10 V/m, 5 V/m, 4 V/m, 3V/m, 2 V/m, 1 V/m, or less.

The systems and methods described with respect to FIGS. 2A-B may be usedto isolate cell beads from particles unoccupied with biologicalparticles, and/or separate singularly occupied cell beads fromunoccupied and multiply occupied cell beads. As described elsewhereherein, a plurality of particles may comprise a first subset ofparticles (e.g., cell beads) occupied by biological particles (e.g.,cells) and a second subset of particles unoccupied by biologicalparticles. Both occupied and unoccupied particles may comprisefield-attractable particles. Occupied particles may include singularlyoccupied cell beads, each containing a single biological particle, andmultiply occupied cell beads, each containing two or more biologicalparticles. For example, such particles comprising the field-attractableparticles may be generated from polymerizing the plurality of dropletscomprising the field-attractable particles (e.g., in FIG. 1). In achannel structure including channel segments 202, 204, and 206 meetingat a channel intersection 211, the plurality of particles may bedirected to flow (e.g., as suspensions in a fluid, e.g., aqueous fluid)to the channel intersection 211 for subsequent sorting. A controller 220can be operatively coupled to a fluid flow unit 218, to facilitate flowof fluid in the channel structure, and a field application unit 216, toapply one or more fields to the channel structure.

In operation, a plurality of discrete particles, including both cellbeads and unoccupied particles, can be directed to flow along channelsegment 202 into intersection 211. The plurality of particles cancomprise a first subset of particles (e.g., cell beads) that are eachoccupied with at least a biological particle and a second subset ofparticles that are each unoccupied. Every particle, including cell beadsand unoccupied particles, can comprise some concentration offield-attractable particles. As described with respect to the relativeconcentrations of field-attractable particles in occupied and unoccupieddroplets, a given unoccupied particle can have a higher concentration offield-attractable particles than a given cell bead (e.g., a givenoccupied particle) to account for the volume occupied by a biologicalparticle in a cell bead.

After sorting at or near the intersection 211, the first subset ofparticles (e.g., cell beads) can be directed to flow along channelsegment 206 and away from the intersection 211, and the second subset ofparticles can be directed to flow along channel segment 204 and awayfrom the intersection 211.

The fluid flow unit 218 can be configured to subject the second fluid208 containing the plurality of particles, including both cell beads andunoccupied particles, to flow along the channel 202 towards theintersection 211. The fluid flow unit 218 can be configured to subjectthe second fluid 208 containing a plurality of particles, wherein amajority of the particles is unoccupied, to flow along the channel 204away from the intersection 211. The fluid flow unit 218 can beconfigured to subject the second fluid 208 containing a plurality ofparticles, wherein a majority of the particles is cell beads (e.g.,occupied particles), to flow along the channel 206 away from theintersection 211. Alternatively, the fluid flow unit 218 can beconfigured to subject the second fluid 208 containing a plurality ofparticles wherein a majority of the particles is unoccupied particles,to flow along the channel 206 away from the intersection 211, andconfigured to subject the second fluid 208 containing a plurality ofparticles, wherein a majority of the particles is cell beads, to flowalong the channel 204 away from the intersection 211. The fluid flowunit 218 can be operatively coupled to the controller 220. For example,the fluid flow unit 218 may receive instructions from the controller 220regarding fluid pressure and/or velocity.

The field application unit 216 can be configured to apply a force fieldto the channel structure. In some instances, the field application unit216 can be configured to apply a force field at or near the intersection211 such that the second subset of particles (unoccupied particles) isgenerally directed along the channel segment 204 and away from theintersection 211, and the first subset of particles (cell beads) isgenerally directed along the channel segment 206 and away from theintersection 211, thereby isolating the two subsets of particles.

For example, the field application unit 216 can apply a magnetic fieldat or near the intersection 211. The field application unit 216 can be amagnet and/or a circuit (e.g., current carrying device) configured togenerate a magnetic field. On account of each particle containingfield-attractable particles (e.g., paramagnetic particles), eachparticle may be attracted (e.g., due to paramagnetic particles) orrepelled (e.g., due to diamagnetic particles) to or away, respectively,from the magnetic field. The degree of attraction (or repulsion) can beproportional to a number (and/or a concentration) of field-attractableparticles in each particle. That is, the magnetic force acting on aparticle, from the same magnetic field, can be proportional to a number(and/or a concentration) of field-attractable particles in the particle.For example, assuming that (i) the particle is spherical and has theradius R_(CB), (ii) a biological particle is spherical and has theradius R₊, and (iii) the concentration of field-attractable particles inthe particle is substantially uniform, the ratio of a number offield-attractable particles in a singularly occupied cell bead (N₊)(wherein the cell bead contains a single biological particle) to anumber of field-attractable particles in an unoccupied particle (N⁻)will be, and thus the ratio of a magnetic force acting on a singularlyoccupied cell bead (F_(M+)) to a magnetic force acting on an unoccupiedparticle (F_(M−)) will be:

$\frac{N_{+}}{N_{-}} = {\frac{F_{M +}}{F_{M -}} = {1 - \left( \frac{R_{+}}{R_{CB}} \right)^{3}}}$

That is, there may be a stronger (differential) force acting on a givenunoccupied particle than a given cell bead. As can be appreciated, theabove ratio may change with deviations from the above assumptions (e.g.,non-spherical biological particle, non-spherical particle, non-uniformconcentration of field-attractable particles in volume of particle,etc.).

In another example, the field application unit 216 can apply an electricfield at or near the intersection 211. On account of each particlecontaining field-attractable particles (e.g., conductive particles),each particle may be attracted or repelled to or away, respectively,from the electric field. The degree of attraction (or repulsion) can beproportional to a number (and/or a concentration) of field-attractableparticles in each particle. That is, the electric force acting on aparticle, from the same electric field, can be proportional to a number(and/or a concentration) of field-attractable particles in the particle.As previously described above, assuming that (i) the particle isspherical and has the radius R₀₃, (ii) a biological particle isspherical and has the radius R₊, and (iii) the concentration offield-attractable particles in the volume of a particle is substantiallyuniform, the ratio of a number of field-attractable particles in asingularly occupied cell bead (N₊) (wherein the cell bead contains asingle biological particle) to a number of field-attractable particlesin an unoccupied particle (N) will be, and thus the ratio of an electricforce acting on a singularly occupied cell bead (F_(E+)) to an electricforce acting on an unoccupied particle (F_(E)) will be:

$\frac{N_{+}}{N_{-}} = {\frac{F_{E +}}{F_{E -}} = {1 - \left( \frac{R_{+}}{R_{D}} \right)^{3}}}$

As can be appreciated, the above ratio may change with deviations fromthe above assumptions (e.g., non-spherical biological particle,non-spherical particle, non-uniform concentration of field-attractableparticles in volume of a particle, etc.). In some instances, the fluidflow unit 218 can apply both an electric field and a magnetic field.

The field application unit 216 can be operatively coupled to thecontroller 220. For example, the field application unit 216 may receiveinstructions from the controller 220 regarding force field strength,orientation, frequency, and/or other variables. The controller 220 mayinstruct the field application unit 216 to apply a force fieldsufficiently strong and in a sufficiently targeted direction towards themixed (occupied and unoccupied) particles such as to direct theunoccupied particles in one channel and direct the occupied particles toanother channel. In an example, the field application unit 216 can beplaced in a location closer to a first channel (e.g., channel 204) thana second channel (e.g., channel 206) to direct the unoccupied particles(which are subject to a stronger force from the same field) to the firstchannel, assuming that the field is strongest when closest to the fieldapplication unit 216. The stronger a force from the field acts on aparticle, the more likely that the particle will deviate from an initialflow direction (e.g., direction of flow in channel 202) into anotherchannel having another direction. In some instances, the fieldapplication unit may be located at least in part downstream, from theintersection 211, of a channel intended to isolate unoccupied particles(e.g., channel 204).

For example, a force field applied can be strong enough to direct theunoccupied particles to flow to a first channel but weak enough todirect (or leave be) the occupied particles to flow to a second channel.In some instances, a magnetic field applied by the field applicationunit 216 can have a magnetic flux density range from at least about 10⁻⁵Teslas (T) to about 1 T. Alternatively, the magnetic flux density can beless than or equal to about 10⁻⁵ T and/or greater than or equal to about1 T. In some instances, an electric field applied by the fieldapplication unit 216 can have an electric field strength of at leastabout 1 volt per meter (V/m), 2 V/m, 3 V/m, 4 V/m, 5 V/m, 10 V/m, ormore. Alternatively, the electric field strength can be less than about10 V/m, 5 V/m, 4 V/m, 3 V/m, 2 V/m, 1 V/m, or less.

Similarly, singularly occupied cell beads may be sorted from unoccupiedparticles and multiply occupied cell beads by using the systems andmethods described with respect to FIG. 2B but introducing a plurality ofparticles (comprising a first subset of singularly occupied cell beads,a second subset of unoccupied particles, and a third subset of multiplyoccupied cell beads), each particle comprising field attractableparticles, in place of the plurality of droplets comprising the fieldattractable particles.

While FIG. 2A and FIG. 2B each depicts a channel structure wherein asecond channel (e.g., channel 204) branches off a first channel (e.g.,channel 202) at a sorting intersection (e.g., intersection 211) suchthat a third channel (channel 204) continues in the same direction asthe first channel, it can be appreciated that the systems and methodsdisclosed herein may be application to different channel structures. Forexample, the third channel can be at a different angle (e.g., not) 180°than the first channel. In some examples, the channel structure may havemore than two channels branching off the first channel, wherein thefield application unit 216 is configured to separate the droplets intounoccupied droplets, droplets containing only one biological particle,droplets containing more than one biological particles, dropletscontaining only barcode carrying beads, droplets containing both abiological particle and barcode carrying beads, or other variations. Insome examples, the channel structure may have more than two channelsbranching off the first channel, wherein the field application unit 216is configured to separate the particles into unoccupied particles,particles containing only one biological particle, particles containingmore than one biological particles, particles containing only barcodecarrying beads, particles containing both a biological particle andbarcode carrying beads, or other variations. For example, the morevolume of a droplet or a particle is occupied by one or more biologicalparticles and/or one or more barcode carrying beads contained therein,the weaker can be the force acting on the droplet or the particle by thefield applied by the field application unit 216, and thus the less thedeviation in direction of flow relative to the direction of flow in thefirst channel. That is, upon application of a force field, theunoccupied droplets or particles may be capable of deviating the most(e.g., to a channel closest to the field application unit), a droplet orparticle containing a single biological particle (e.g., singularlyoccupied cell bead) may be capable of deviating but not as much as theunoccupied droplets or particles (e.g., to a channel second closest tothe field application unit), and a droplet or particle containing both abiological particle and a barcode carrying bead may be capable ofdeviating the least of the three types of droplets or particles (e.g.,to a channel farthest from the field application unit).

While FIGS. 2A-2B depict narrow channels allowing for the flow ofdroplets and/or particles only in single file, the systems and methodsdisclosed herein may be applicable to channels having a broader width(e.g., diameter) that allows for the flow of droplets and/or particlesin more than single file.

While FIG. 2A depicts one controller 220 operatively coupled to both thefluid flow unit 218 and the field application unit 216, a separatecontroller can be coupled to the fluid flow unit 218 and a separatecontroller can be coupled to the field application unit 216. The twoseparate controllers may or may not be in communication with each other.In some instances, there may be a plurality of controllers (e.g., twocontrollers to a fluid flow unit 218), wherein each controller may ormay not be in communication with each other. The controller 220 may sendinstructions to the fluid flow unit 218 and/or the field applicationunit 216 via wired connection and/or wireless connection (e.g., Wi-Fi,Bluetooth, NFC, etc.).

FIG. 3 shows another example of a microfluidic channel structure forseparating occupied droplets from unoccupied droplets. As describedelsewhere herein, when droplets are generated, there may be a firstsubset population of occupied droplets containing one or more biologicalparticles and a second subset population of unoccupied droplets notcontaining any biological particles. In some cases, the droplets mayadditionally contain one or more barcode carrying beads. For example, adroplet may have only a biological particle, a droplet may have only abarcode carrying bead, a droplet may have both a biological particle anda barcode carrying bead, or a droplet may have neither biologicalparticles nor barcode carrying beads. In some cases, the majority ofoccupied partitions can include no more than one biological particle peroccupied partition and, in some cases, some of the generated partitionscan be unoccupied (of any biological particle). In some cases, though,some of the occupied partitions may include more than one biologicalparticle. In some cases, the partitioning process may be controlled suchthat fewer than about 25% of the occupied partitions contain more thanone biological particle, and in many cases, fewer than about 20% of theoccupied partitions have more than one biological particle, while insome cases, fewer than about 10% or even fewer than about 5% of theoccupied partitions include more than one biological particle perpartition.

As shown in FIG. 3, the channel structure can include channel segments302, 304, and 306 meeting at a channel intersection 311. In someinstances, the outflow channel 108 of the emulsion carrying thegenerated droplets in FIG. 1 can be upstream of the channel segment 302,such that the generated droplets are directed to flow to the channelintersection 211 for subsequent sorting. A controller 322 can beoperatively coupled to a fluid flow unit 318, to facilitate flow offluid in the channel structure, a sensor 320, to detect at least acharacteristic of a droplet or a plurality of droplets, and a pressureapplication unit 316, to apply a pressure pulse to the channelstructure.

In operation, a plurality of discrete droplets, each comprising a firstaqueous fluid 310 can flow as emulsions in a second fluid 308, whereinthe second fluid 308 is immiscible to the first aqueous fluid 310. Thedroplets being transported along channel segment 302 into intersection311 can comprise a first subset of droplets 314 that are each occupiedwith at least a biological particle and/or a barcode carrying bead and asecond subset of droplets 312 that are each unoccupied. Every droplet,including occupied and unoccupied droplets, may or may not comprise someconcentration of field-attractable particles. Although FIG. 3 depictseach droplet as comprising field-attractable particles 315, this is notrequired.

After sorting at or near the intersection 311, the first subset ofdroplets 314 can be directed to flow along channel segment 306 and awayfrom the intersection 311, and the second subset of droplets 312 can bedirected to flow along channel segment 304 and away from theintersection 311.

The fluid flow unit 318 can be configured to subject the second fluid308 containing a plurality of droplets, including both occupied dropletsand unoccupied droplets, to flow along the channel 302 towards theintersection 311. The fluid flow unit 318 can be configured to subjectthe second fluid 308 containing a plurality of droplets, wherein amajority of the droplets is unoccupied droplets, to flow along thechannel 304 away from the intersection 311. The fluid flow unit 318 canbe configured to subject the second fluid 308 containing a plurality ofdroplets, wherein a majority of the droplets is occupied droplets, toflow along the channel 306 away from the intersection 311.Alternatively, the fluid flow unit 318 can be configured to subject thesecond fluid 308 containing a plurality of droplets, wherein a majorityof the droplets is unoccupied droplets, to flow along the channel 306away from the intersection 311, and configured to subject the secondfluid 308 containing a plurality of droplets, wherein a majority of thedroplets is occupied droplets, to flow along the channel 304 away fromthe intersection 311. The fluid flow unit 318 can be operatively coupledto the controller 322. For example, the fluid flow unit 318 may receiveinstructions from the controller 322 regarding fluid pressure and/orvelocity.

In some instances, the fluid flow unit 318 may comprise a compressor toprovide positive pressure at an upstream location to direct the fluidfrom the upstream location to flow to a downstream location. In someinstances, the fluid flow unit 318 may comprise a pump to providenegative pressure at a downstream location to direct the fluid from anupstream location to flow to the downstream location. In some instances,the fluid flow unit 318 may comprise both a compressor and a pump, eachat different locations. In some instances, the fluid flow unit 318 maycomprise different devices at different locations. The fluid flow unit318 may comprise an actuator. While FIG. 3 depicts one fluid flow unit318, it may be appreciated that there may be a plurality of fluid flowunits 318, each in communication with the controller 322 and/or witheach other. For example, there can be a separate fluid flow unit todirect the fluid in channel 302 towards the intersection 311, a separatefluid flow unit to direct the fluid in channel 304 away from theintersection 311, and a separate fluid flow unit to direct the fluid inchannel 306 away from the intersection 311.

The sensor 320 can be configured to sense at least a characteristic of adroplet or a plurality of droplets in the first channel segment 302. Insome instances, the sensor 320 may detect the characteristic of adroplet as the droplet passes the sensor 320. The sensor 320 may belocated upstream of the intersection 311. One or more characteristicsdetected by the sensor 320 of a droplet can be indicative of the type ofdroplet, such as whether the droplet is occupied or unoccupied, orwhether the droplet contains a biological particle and/or a barcodecarrying bead. In some instances, the sensor 320 can be an impedancesensor configured to measure bulk impedance when droplets pass by thesensor 320. In some instances, a higher impedance can be measured foroccupied droplets than for unoccupied droplets (e.g., due to mass and/orweight distribution of occupied droplet, etc.). In some instances, thesensor 320 can be an optical sensor configured to measure opticalproperties of a droplet, such as to distinguish whether the droplet isoccupied or unoccupied. The optical sensor and/or a supporting devicemay be configured to emit a detection signal configured to probe one ormore droplets, including for example an electromagnetic signal (e.g., inany wavelength) and/or an acoustic signal. In some instances, theoptical sensor and/or a supporting device may comprise an illuminationsource configured to illuminate the droplet or droplets with one or moretypes of electromagnetic radiation. In some instances, theelectromagnetic radiation can include illumination in one or more of thevisible spectrum, infrared spectrum, the ultraviolet spectrum, andionizing radiation spectrum. In some instances, the ionizing radiationcan include x-rays. Alternatively the sensor 320 may be one or moredevices that are configured to provide one or more of optical sensing,thermal sensing, laser imaging, infrared imaging, capacitance sensing,mass sensing, vibration sensing across at least a portion of theelectromagnetic spectrum, and magnetic induction sensing. The sensor 320can be operatively coupled to the controller 322. For example, thesensor 320 may transmit sensor data (e.g., on one or morecharacteristics of a droplet or a plurality of droplets) to thecontroller 322. The controller 322 may then use such data to determinewhether the droplet is occupied or unoccupied.

While FIG. 3 depicts one sensor 320, it may be appreciated that theremay be a plurality of sensors, each in communication with the controller322 and/or with each other. For example, there can be a plurality ofsensors upstream of the intersection 311 at different locations, forexample, detecting one or more characteristics of a droplet or aplurality of droplets at different angles.

The pressure application unit 316 can be configured to apply a pressurepulse to the channel structure. In some instances, the pressureapplication unit 316 can be configured to apply a pressure pulse at ornear the intersection 311 such that, via hydrodynamic forces, the secondsubset of droplets (unoccupied droplets) are generally directed alongthe channel segment 304 and away from the intersection 311, and thefirst subset of droplets (occupied droplets) are generally directedalong the channel segment 306 and away from the intersection 311,thereby isolating the two subsets of droplets.

In some instances, the pressure application unit 316 may comprise acompressor to provide positive pressure pulses at an upstream locationto direct the fluid from the upstream location to flow to a downstreamlocation. In some instances, the pressure application unit 316 maycomprise a pump to provide negative pressure pulses at a downstreamlocation to direct the fluid from an upstream location to flow to thedownstream location. In some instances, the pressure application unit316 may comprise both a compressor and a pump, each at differentlocations. In some instances, the pressure application unit 316 maycomprise different devices at different locations. The pressureapplication unit 316 may comprise an actuator. While FIG. 3 depicts onepressure application unit 316, it may be appreciated that there may be aplurality of pressure application units, each in communication with thecontroller 322 and/or with each other. In some instances, the pressureapplication unit 316 and a fluid flow unit 318 may be the same device orsame devices. In some instances, the pressure application unit 316 maybe, entirely or at least in part, external to the microfluidic structure(e.g., microfluidic channels), as illustrated in FIG. 3. In someinstances, the pressure application unit 316 may be, entirely or atleast in part, internal to and/or integral to the microfluidicstructure. For example, a pressure pulse may be generated by deflectionof membranes. In some instances, a pressure pulse may be generated fromgeneration of air bubbles, wherein expansion of the bubble may displacefluid parcels.

Occupied droplets and unoccupied droplets may respond differently to apressure pulse, for example due to varying predetermined particle andfluid characteristics. In some instances, singularly occupied dropletsand multiply occupied droplets may respond differently to a pressurepulse. The predetermined particle and fluid characteristics can includesize of a droplet, mass of a droplet, viscosity of droplet suspension inthe emulsion, deformability, and other characteristics. That is, a givensingularly occupied droplet, a multiply occupied droplet, and anunoccupied droplet may respond differently when subject to hydrodynamicforces triggered by the pressure pulses. In some instances, thecontroller 322 may, based on a determination made from data receivedfrom the sensor 320 (e.g., determination on whether droplet is occupiedor unoccupied), instruct the pressure application unit 316 to applydifferent pressure pulses, for example, by varying frequency of thepulses and/or changing pressure differential. For example, the pressureapplication unit 316 may apply a first type of pressure pulse when anoccupied droplet is approaching the intersection 311 and a second typeof pressure pulse when an unoccupied droplet is approaching theintersection 311. Alternatively, the same pressure pulse can be appliedfor any type of droplet approaching the intersection 311, and thedroplet may react (or respond) differently (e.g., deviating from a fluidflow direction at different angles) depending on whether the droplet isoccupied or unoccupied.

The pressure application unit 316 can be operatively coupled to thecontroller 320. For example, the pressure application unit 316 mayreceive instructions from the controller 322 regarding pressure pulsestrength, frequency, and/or other variables.

The systems and methods described with respect to FIG. 3 may be used toseparate occupied particles from unoccupied particles. As describedelsewhere herein, a plurality of particles may comprise a first subsetof particles (e.g., cell beads) occupied by biological particles (e.g.,cells) and a second subset of particles unoccupied by biologicalparticles. In a channel structure including channel segments 302, 304,and 306 meeting at a channel intersection 311, the plurality ofparticles may be directed to flow (e.g., as suspensions in a fluid,e.g., aqueous fluid) to the channel intersection 311 for subsequentsorting. A controller 322 can be operatively coupled to a fluid flowunit 318, to facilitate flow of fluid in the channel structure, a sensor320, to detect at least a characteristic of a particle or a plurality ofparticles, and a pressure application unit 316, to apply a pressurepulse to the channel structure.

After sorting at or near the intersection 311, the first subset ofparticles (e.g., cell beads) can be directed to flow along channelsegment 306 and away from the intersection 311, and the second subset ofparticles can be directed to flow along channel segment 304 and awayfrom the intersection 311.

The fluid flow unit 318 can be configured to subject the second fluid308 containing a plurality of particles, including both cell beads andunoccupied particles, to flow along the channel 302 towards theintersection 311. The fluid flow unit 318 can be configured to subjectthe second fluid 308 containing a plurality of particles, wherein amajority of the particles is unoccupied particles, to flow along thechannel 304 away from the intersection 311. The fluid flow unit 318 canbe configured to subject the second fluid 308 containing a plurality ofparticles, wherein a majority of the particles is occupied particles, toflow along the channel 306 away from the intersection 311.Alternatively, the fluid flow unit 318 can be configured to subject thesecond fluid 308 containing a plurality of particles, wherein a majorityof the particles is unoccupied particles, to flow along the channel 306away from the intersection 311, and configured to subject the secondfluid 308 containing a plurality of particles, wherein a majority of theparticles is occupied particles, to flow along the channel 304 away fromthe intersection 311. The fluid flow unit 318 can be operatively coupledto the controller 322. For example, the fluid flow unit 318 may receiveinstructions from the controller 322 regarding fluid pressure and/orvelocity.

The sensor 320 can be configured to sense at least a characteristic of aparticle or a plurality of particles in the first channel segment 302.In some instances, the sensor 320 may detect the characteristic of aparticle as the particle passes the sensor 320. The sensor 320 may belocated upstream of the intersection 311. One or more characteristicsdetected by the sensor 320 of a particle can be indicative of the typeof particle, such as whether the particle is occupied or unoccupied, orwhether the particle contains a biological particle and/or a barcodecarrying bead. In some instances, the sensor 320 can be an impedancesensor configured to measure bulk impedance when particles pass by thesensor 320. In some instances, a higher impedance can be measured foroccupied particles than for unoccupied particles (e.g., due to massand/or weight distribution of occupied particle, etc.). In someinstances, the sensor 320 can be an optical sensor configured to measureoptical properties of a particle, such as to distinguish whether theparticle is occupied or unoccupied. The optical sensor and/or asupporting device may be configured to emit a detection signalconfigured to probe one or more particles, including for example anelectromagnetic signal (e.g., in any wavelength) and/or an acousticsignal. In some instances, the optical sensor and/or a supporting devicemay comprise an illumination source configured to illuminate theparticle or particles with one or more types of electromagneticradiation. In some instances, the electromagnetic radiation can includeillumination in one or more of the visible spectrum, infrared spectrum,the ultraviolet spectrum, and ionizing radiation spectrum. In someinstances, the ionizing radiation can include x-rays. Alternatively thesensor 320 may be one or more devices that are configured to provide oneor more of optical sensing, thermal sensing, laser imaging, infraredimaging, capacitance sensing, mass sensing, vibration sensing across atleast a portion of the electromagnetic spectrum, and magnetic inductionsensing. The sensor 320 can be operatively coupled to the controller322. For example, the sensor 320 may transmit sensor data (e.g., on oneor more characteristics of a particle or a plurality of particles) tothe controller 322. The controller 322 may then use such data todetermine whether the particle is occupied or unoccupied.

While FIG. 3 depicts one sensor 320, it may be appreciated that theremay be a plurality of sensors, each in communication with the controller322 and/or with each other. For example, there can be a plurality ofsensors upstream of the intersection 311 at different locations, forexample, detecting one or more characteristics of a particle or aplurality of particles at different angles.

The pressure application unit 316 can be configured to apply a pressurepulse to the channel structure. In some instances, the pressureapplication unit 316 can be configured to apply a pressure pulse at ornear the intersection 311 such that, via hydrodynamic forces, the secondsubset of particles (unoccupied particles) are generally directed alongthe channel segment 304 and away from the intersection 311, and thefirst subset of particles (cell beads) are generally directed along thechannel segment 306 and away from the intersection 311, therebyisolating the two subsets of particles.

Occupied particles and unoccupied particles may respond differently to apressure pulse, for example due to varying predetermined particle andfluid characteristics. The predetermined particle and fluidcharacteristics can include size of a particle, mass of a particle,viscosity of particle suspension in the fluid, deformability, and othercharacteristics. That is, a given singularly occupied cell bead,multiply occupied cell bead, and an unoccupied particle may responddifferently when subject to hydrodynamic forces triggered by thepressure pulses. In some instances, the controller 322 may, based on adetermination made from data received from the sensor 320 (e.g.,determination on whether particle is occupied or unoccupied), instructthe pressure application unit 316 to apply different pressure pulses,for example, by varying frequency of the pulses and/or changing pressuredifferential. For example, the pressure application unit 316 may apply afirst type of pressure pulse when an occupied particle is approachingthe intersection 311 and a second type of pressure pulse when anunoccupied particle is approaching the intersection 311. Alternatively,the same pressure pulse can be applied for any type of particleapproaching the intersection 311, and the particle may react (orrespond) differently (e.g., deviating from a fluid flow direction atdifferent angles) depending on whether the particle is occupied orunoccupied. In some instances, different pressure pulses may be appliedas between occupied and unoccupied droplets, occupied and unoccupiedparticles, singularly occupied and multiply occupied droplets, and/orsingularly occupied and multiply occupied cell beads.

The pressure application unit 316 can be operatively coupled to thecontroller 320. For example, the pressure application unit 316 mayreceive instructions from the controller 322 regarding pressure pulsestrength, frequency, and/or other variables.

While FIG. 3 depicts a channel structure wherein a second channel(channel 304) branches off a first channel (channel 302) such that athird channel (channel 304) continues in the same direction as the firstchannel, it can be appreciated that the systems and methods disclosedherein may be application to different channel structures. For example,the third channel can be at a different angle (e.g., not 180°) than thefirst channel. In some examples, the channel structure may have morethan two channels branching off the first channel, wherein the pressureapplication unit 316 is configured to separate the droplets intounoccupied droplets, droplets containing only one biological particle,droplets containing more than one biological particles, dropletscontaining only barcode carrying beads, droplets containing both abiological particle and barcode carrying beads, or other variations. Thepressure application unit 316 can be configured to separate particlesinto unoccupied particles, particles containing only one biologicalparticle, particles containing more than one biological particle,particles containing only barcode carrying beads, particles containingboth a biological particle and barcode carrying beads, or othervariations. For example, upon application of a pressure pulse, theunoccupied droplets or particles may be capable of deviating the most(e.g., to a first channel), a droplet or particle containing a singlebiological particle may be capable of deviating but not as much as theunoccupied droplets or particles (e.g., to a second channel), and adroplet or particle containing both a biological particle and a barcodecarrying bead may be capable of deviating the least of the three typesof droplets or particles (e.g., to a third channel).

While FIG. 3 depicts narrow channels allowing for the flow of dropletsor particles only in single file, the systems and methods disclosedherein may be applicable to channels having a broader width (e.g.,diameter) that allows for the flow of droplets or particles in more thansingle file.

While FIG. 3 depicts one controller 322 operatively coupled to all ofthe fluid flow unit 318, the sensor 320, and the pressure applicationunit 316, a separate controller can be coupled to the fluid flow unit318, a separate controller can be coupled to the sensor 320, and aseparate controller can be coupled to the pressure application unit 216.The three separate controllers may or may not be in communication witheach other. In some instances, there may be a plurality of controllers(e.g., two controllers to a fluid flow unit 318), wherein eachcontroller may or may not be in communication with each other. Thecontroller 322 may send instructions to the fluid flow unit 318, sensor320, and/or the pressure application unit 316 via wired connectionand/or wireless connection (e.g., Wi-Fi, Bluetooth, NFC, etc.). Thecontroller 322 may receive data from the fluid flow unit 318, sensor320, and/or the pressure application unit 316 via wired connectionand/or wireless connection (e.g., Wi-Fi, Bluetooth, NFC, etc.). In someinstances, the components can be directly or indirectly be incommunication with each other, with or without going through thecontroller 322. For example, the sensor 320 may be directly coupled tothe pressure application unit 316.

The separation systems and methods disclosed herein may achieve superPoisson loading. For example, the droplets can be separated into twosubsets such that at least about 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%,60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or greater of a firstsubset of droplets that is isolated are occupied droplets (e.g.,containing at least one biological particle). Such occupancy may begreater than or equal to 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%,60%, 70%, 80%, 90%, 95%, or higher. Alternatively, less than about 97%of the first subset of droplets can be occupied droplets. In someinstances, at least about 97%, 98%, 99%, or a higher percentage of asecond subset of droplets that is isolated can be unoccupied droplets(e.g., not containing any biological particle and not containing anybarcode carrying beads). Alternatively, less than about 97% of thesecond subset of droplets can be unoccupied droplets. For example, theparticles can be separated into two subsets such that at least about 5%,10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%,98%, 99%, or greater of a first subset of particles that is isolated arecell beads (e.g., containing at least one biological particle). Suchoccupancy may be greater than or equal to 1%, 2%, 3%, 4%, 5%, 10%, 20%,30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or higher. Alternatively, lessthan about 97% of the first subset of particles can be occupiedparticles (e.g., cell beads). In some instances, at least about 97%,98%, 99%, or a higher percentage of a second subset of particles that isisolated can be unoccupied particles (e.g., not containing anybiological particle and not containing any barcode carrying beads).Alternatively, less than about 97% of the second subset of particles canbe unoccupied particles.

The channel networks, e.g., as described herein above or further below,can be fluidly coupled to appropriate fluidic components. For example,the inlet channel segments (e.g., channel segment 202 in FIG. 2A,channel segments 102, 104, 106 in FIG. 1) are fluidly coupled toappropriate sources of the materials they are to deliver to a channeljunction (e.g., intersection 211 in FIG. 2A, junction 110 in FIG. 1).For example, channel segment 202 will be fluidly coupled to a source ofan aqueous suspension of biological particles (e.g., biologicalparticles 114 in FIG. 1) to be analyzed and field-attractable particles(e.g., field-attractable particles 115 in FIG. 1). Channel segments 104and 106 may then be fluidly connected to one or more sources of thenon-aqueous (or other immiscible) fluid. These sources may include anyof a variety of different fluidic components, from simple reservoirsdefined in or connected to a body structure of a microfluidic device, tofluid conduits that deliver fluids from off-device sources, manifolds,fluid flow units (e.g., actuators, pumps, compressors) or the like.Likewise, the outlet channel segment 206 may be fluidly coupled to areceiving vessel or conduit for the partitioned cells for subsequentprocessing. Again, this may be a reservoir defined in the body of amicrofluidic device, or it may be a fluidic conduit for delivering thepartitioned cells to a subsequent process operation, instrument orcomponent.

In some instances, a plurality of droplets not containing anyfield-attractable particles, may also be sorted using dielectrophoresis.For example, occupied droplets and unoccupied droplets can havedifferent dielectric properties. When an electric field is applied, suchas via methods described elsewhere herein (e.g., with reference to FIG.2A), to a plurality of droplets comprising both occupied droplets andunoccupied droplets, such as at an intersection wherein a first channelbranches off into a second channel and a third channel, the occupieddroplets may be directed to flow through the first channel andunoccupied droplets may be directed to flow through the second channel,due at least in part to the varying interactions with (or influence of)the electric field of the occupied droplets and the unoccupied dropletshaving different dielectric properties. Such systems and methods fordielectrophoresis may also be used to sort a plurality of particles intoa first subset of occupied particles (e.g., cell beads) and a secondsubset of unoccupied particles.

The biological particle can be subjected to conditions sufficient topolymerize or gel the precursors. The conditions sufficient topolymerize or gel the precursors may comprise exposure to heating,cooling, electromagnetic radiation, or light. The conditions sufficientto polymerize or gel the precursors may comprise any conditionssufficient to polymerize or gel the precursors. Following polymerizationor gelling, a polymer or gel may be formed around the biologicalparticle. The polymer or gel may be diffusively permeable to chemical orbiochemical reagents. The polymer or gel may be diffusively impermeableto macromolecular constituents of the biological particle. In thismanner, the polymer or gel may act to allow the biological particle tobe subjected to chemical or biochemical operations while spatiallyconfining the macromolecular constituents to a region of the dropletdefined by the polymer or gel. The polymer or gel may include one ormore of disulfide cross-linked polyacrylamide, agarose, alginate,polyvinyl alcohol, polyethylene glycol (PEG)-diacrylate, PEG-acrylate,PEG-thiol, PEG-azide, PEG-alkyne, other acrylates, chitosan, hyaluronicacid, collagen, fibrin, gelatin, or elastin. The polymer or gel maycomprise any other polymer or gel.

The polymer or gel may be functionalized to bind to targeted analytes,such as nucleic acids, proteins, or other analytes. The polymer or gelmay be polymerized or gelled via a passive mechanism. The polymer or gelmay be stable in alkaline conditions or at elevated temperature. Thepolymer or gel may have mechanical properties similar to the mechanicalproperties of the bead. For instance, the polymer or gel may be of asimilar size to the bead. The polymer or gel may have a mechanicalstrength (e.g. tensile strength) similar to that of the bead. Thepolymer or gel may be of a lower density than an oil. The polymer or gelmay be of a density that is roughly similar to that of a buffer. Thepolymer or gel may have a tunable pore size. The pore size may be chosento, for instance, retain denatured nucleic acids. The pore size may bechosen to maintain diffusive permeability to exogenous chemicals such assodium hydroxide (NaOH) and/or endogenous chemicals such as inhibitors.The polymer or gel may be biocompatible. The polymer or gel may maintainor enhance cell viability. The polymer or gel may be biochemicallycompatible. The polymer or gel may be polymerized and/or depolymerizedthermally, chemically, enzymatically, and/or optically.

The polymer may comprise poly(acrylamide-co-acrylic acid) crosslinkedwith disulfide linkages. The preparation of the polymer may comprise atwo-step reaction. In the first activation step,poly(acrylamide-co-acrylic acid) may be exposed to an acylating agent toconvert carboxylic acids to esters. For instance, thepoly(acrylamide-co-acrylic acid) may be exposed to4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride(DMTMM). The polyacrylamide-co-acrylic acid may be exposed to othersalts of 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium. Inthe second cross-linking step, the ester formed in the first step may beexposed to a disulfide crosslinking agent. For instance, the ester maybe exposed to cystamine (2,2′-dithiobis(ethylamine)). Following the twosteps, the biological particle may be surrounded by polyacrylamidestrands linked together by disulfide bridges. In this manner, thebiological particle may be encased inside of or comprise a gel or matrix(e.g., polymer matrix) to form a “cell bead.” A cell bead can containbiological particles (e.g., a cell) or macromolecular constituents(e.g., RNA, DNA, proteins, etc.) of biological particles. A cell beadmay include a single cell or multiple cells, or a derivative of thesingle cell or multiple cells. For example after lysing and washing thecells, inhibitory components from cell lysates can be washed away andthe macromolecular constituents can be bound as cell beads. Systems andmethods disclosed herein can be applicable to both cell beads (and/ordroplets or other partitions) containing biological particles and cellbeads (and/or droplets or other partitions) containing macromolecularconstituents of biological particles. In some cases, the cell bead mayfurther comprise one or more field-attractable particles (e.g.,paramagnetic particles, conductive particles, etc.), such as via thesystems and methods described elsewhere herein, for facilitatingsubsequent sorting and/or solvent exchange. The field-attractableparticles may be trapped in the gel matrix. In some instances, thefield-attractable particles may be trapped evenly throughout the gelmatrix. In some instances, the field-attractable particles may betrapped throughout the gel matrix such as to subject the whole of thecell bead evenly to a force (e.g., magnetic, electric) field.

In another aspect, provided herein are systems and methods for selectivepolymerization of partitions (or cells therein). In some instances, thepartitions may be selectively polymerized based on occupancy. In someinstances, the partitions may be selectively polymerized based on size.

FIG. 4 shows an example of a microfluidic channel structure forselective polymerization of partitions based on occupancy. As describedelsewhere herein, in some cases, the majority of occupied partitions caninclude no more than one biological particle per occupied partition and,in some cases, some of the generated partitions can be unoccupied (ofany biological particle). In some cases, though, some of the occupiedpartitions may include more than one biological particle. In some cases,the partitioning process may be controlled such that fewer than 25% ofthe occupied partitions contain more than one biological particle, andin many cases, fewer than 20% of the occupied partitions have more thanone biological particle, while in some cases, fewer than 10% or evenfewer than 5% of the occupied partitions include more than onebiological particle per partition.

The emulsion mechanism of FIG. 4 can largely parallel that of FIG. 1. Asshown in FIG. 4, the channel structure can include channel segments 401,402, 404, 406 and 408. Channel segments 401 and 402 can communicate at achannel junction 409. Channel segments 402, 404, 406, and 408 cancommunicate at a channel junction 410. In operation, a first aqueousfluid 412 can be delivered to junction 409 from each of channel segments402 and 401. Cells 414 can be introduced into the junction 409 via thechannel segment 401 as suspensions in the first aqueous fluid 412flowing along the channel segment 401. The first aqueous fluid 412 mayor may not contain suspended field-attractable particles 115. Asdescribed elsewhere herein, occupied droplets and unoccupied dropletsmay be sorted via the field-attractable particles 115. A second fluid416 that is immiscible with the aqueous fluid 412 is delivered to thejunction 410 from each of channel segments 404 and 406 to creatediscrete droplets 418, 420 of the first aqueous fluid 412 flowing intochannel segment 408, and flowing away from junction 410. A discretedroplet generated may or may not include biological particles 414.

The second fluid 416 can comprise an oil, such as a fluorinated oil, anda surfactant, such as a fluorosurfactant for stabilizing the resultingdroplets, e.g., inhibiting subsequent coalescence of the resultingdroplets. Examples of particularly useful partitioning fluids andfluorosurfactants are described for example, in U.S. Patent ApplicationPublication No. 2010/0105112, which is entirely incorporated herein byreference for all purposes.

The generated droplets may comprise two subsets of droplets: (1)occupied droplets 418, containing one or more biological particles 414,and (2) unoccupied droplets 420, not containing any biological particles414.

A photo polymerization light source 424, such as a laser, can be locateddownstream of the junction 410 to selectively polymerize occupieddroplets (e.g., polymerize the biological particles therein). In someinstances, the light source 424 can be a lamp or light emitting diode(LED). The light source can be an ultraviolet (UV) radiation source. Thelight source 424 can generate optical pulses or an electromagnetic beamat a targeted direction. For example, the light source 424 can beconfigured to only emit electromagnetic waves when an occupied dropletis passing by. The light source 424 can be operatively coupled to acontroller 426. The light source 424 can receive instructions from thecontroller 426 on whether or not a droplet passing by the light source424, or a droplet expected to pass by the light source at a certaintime, is occupied. While FIG. 4 depicts one light source 424, it may beappreciated that there may be a plurality of light sources, each incommunication with the controller 426 and/or with each other. Forexample, a plurality of light sources may each be located at differentlocations, including different upstream/downstream locations in thefluidic channels. Alternatively, a different polymerization applicationunit can be used to subject the droplets to a stimulus, such as totrigger polymerization via heating, cooling, electromagnetic radiation,and/or light. The stimulus can be a chemical stimulus. In someinstances, a plurality of the same or different types of polymerizationapplication units can be used at different locations.

A sensor 422 can be configured to sense the presence of a biologicalparticle (or cell) 414 in the fluid flow. For example, the sensor 422can be configured to sense the presence of a biological particle (orcell) 414 in the channel segment 401 upstream of the junction 409. Thesensor 422 may be located upstream of the junction 409. In someinstances, the sensor 422 can be an impedance sensor configured tomeasure bulk impedance when cells 414 pass by the sensor 422. In someinstances, a higher impedance can be measured when a cell 414 passes bythan when only the first aqueous fluid 412 passes by. In some instances,the sensor 422 can be an optical sensor configured to measure opticalproperties of a cell 414. The optical sensor and/or a supporting devicemay be configured to emit a detection signal configured to probe one ormore droplets, including for example an electromagnetic signal (e.g., inany wavelength) and/or an acoustic signal. In some instances, theoptical sensor and/or a supporting device may comprise an illuminationsource configured to illuminate the cell with one or more types ofelectromagnetic radiation. In some instances, the electromagneticradiation can include illumination in one or more of the visiblespectrum, infrared spectrum, the ultraviolet spectrum, and ionizingradiation spectrum. In some instances, the ionizing radiation caninclude x-rays. The illumination can be transillumination orepi-illumination. Alternatively, the sensor 422 may be one or moredevices that are configured to provide one or more of optical sensing,thermal sensing, laser imaging, infrared imaging, capacitance sensing,mass sensing, vibration sensing across at least a portion of theelectromagnetic spectrum, and magnetic induction sensing. The sensor 422may collect sensor data on one or more properties (e.g., opticalproperties, impendence properties, etc.) or characteristics of a cell414.

The sensor 422 can be operatively coupled to the controller 426. Forexample, the sensor 426 may transmit sensor data (e.g., on presence ofone or more cells 414) to the controller 426. The controller 426 maydetermine when a cell has passed by the sensor location from the sensordata. The controller 426 may then use such sensor data, the location ofthe sensor and/or the location in the fluidic channel at which thepresence of a cell 414 was detected, the time the sensor detected apresence of the cell 414 in the flow, fluid flow rate of the firstaqueous fluid 412 in the channel segment 402, fluid flow rate ofemulsion in the channel segment 408, location of the light source 424,time it takes for the sensor 426 to detect and/or transmit data to thecontroller 426, and/or time it takes for the controller 426 to sendinstructions to the light source 424, to send instructions to the lightsource 424 on whether or not to emit an electromagnetic wave topolymerize a droplet. For example, assuming that the fluid flow rates inthe channel segments 402 and/or 408 are substantially constant, thecontroller 426 may determine whether a droplet created (e.g., atjunction 410) at a certain time contains a cell or does not contain acell. Based on the fluid flow rates in the channel segments 402 and/or408, the controller 426 may determine whether a droplet passing by thelight source 424 at a certain time (e.g., time it takes for droplet totravel from junction 410 to the location of the light source 424 site isthe same every time) is occupied or unoccupied.

While FIG. 4 depicts one sensor 422, it may be appreciated that theremay be a plurality of sensors, each in communication with the controller426 and/or with each other. For example, there can be a plurality ofsensors upstream of the junction 409 and/or upstream of the junction 410at different locations, for example, detecting the presence of a cell414.

While FIG. 4 depicts one controller 426 operatively coupled to both thesensor 422 and the light source 424, a separate controller can becoupled to the sensor 422 and a separate controller can be coupled tothe light source 424. The separate controllers may or may not be incommunication with each other. In some instances, there may be aplurality of controllers (e.g., two controllers to sensor 422), whereineach controller may or may not be in communication with each other. Thecontroller 426 may send instructions to sensor 422 and/or the lightsource 424 via wired connection and/or wireless connection (e.g., Wi-Fi,Bluetooth, NFC, etc.). The controller 422 may receive data from thesensor 422 and/or the light source 424 via wired connection and/orwireless connection (e.g., Wi-Fi, Bluetooth, NFC, etc.). In someinstances, the components can be directly or indirectly be incommunication with each other, with or without going through thecontroller 426. For example, the sensor 422 may be directly coupled tothe light source 426.

FIG. 5 shows another example of a microfluidic channel structure forselective polymerization of partitions (e.g., biological particlestherein) based on occupancy.

The emulsion mechanism of FIG. 5 can largely parallel that of FIGS. 1and 4. As shown in FIG. 5, the channel structure can include channelsegments 501, 502, 504, 506 and 508. Channel segments 501 and 502 cancommunicate at a channel junction 509. Channel segments 502, 504, 506,and 508 can communicate at a channel junction 510. In operation, a firstaqueous fluid 512 can be delivered to junction 509 from each of channelsegments 502 and 501. Cells 514 can be introduced into the junction 509via the channel segment 501 as suspensions in the first aqueous fluid512 flowing along the channel segment 501. The first aqueous fluid 512may or may not contain suspended field-attractable particles 515. Asdescribed elsewhere herein, occupied droplets and unoccupied dropletsmay be subsequently sorted via the field-attractable particles 515. Asecond fluid 516 that is immiscible with the aqueous fluid 512 isdelivered to the junction 510 from each of channel segments 504 and 506to create discrete droplets 518, 520 of the first aqueous fluid 512flowing into channel segment 508, and flowing away from junction 510. Adiscrete droplet generated may or may not include biological particles514. Each cell 514 introduced into the droplet can comprise fluorescentlabels (such as in accordance with the widely usedFluorescence-Activated Cell Sorting (FACS) mechanism) or other opticallabels that allow for detection by an optical sensor.

The second fluid 516 can comprise an oil, such as a fluorinated oil,that includes a fluorosurfactant for stabilizing the resulting droplets,e.g., inhibiting subsequent coalescence of the resulting droplets.Examples of particularly useful partitioning fluids andfluorosurfactants are described for example, in U.S. Patent ApplicationPublication No. 2010/0105112, which is entirely incorporated herein byreference for all purposes.

The generated droplets may comprise two subsets of droplets: (1)occupied droplets 518, containing one or more biological particles 514,and (2) unoccupied droplets 520, not containing any biological particles514.

A photo polymerization light source 524, such as a laser, can be locateddownstream of the junction 510 to selectively polymerize occupieddroplets. In some instances, the light source 524 can be a lamp or lightemitting diode (LED). The light source 524 can generate optical pulsesor an electromagnetic beam at a targeted direction. For example, thelight source 524 can be configured to only emit electromagnetic waveswhen an occupied droplet is passing by. The light source 524 can beoperatively coupled to a controller 526. The light source 524 canreceive instructions from the controller 526 on whether or not a dropletpassing by the light source 524, or a droplet expected to pass by thelight source at a certain time, is occupied. While FIG. 5 depicts onelight source 524, it may be appreciated that there may be a plurality oflight sources, each in communication with the controller 526 and/or witheach other. For example, a plurality of light sources may each belocated at different locations, including different upstream/downstreamlocations in the fluidic channels. Alternatively, a differentpolymerization application unit can be used to subject the droplets to astimulus, such as to trigger polymerization of one or more biologicalparticles therein via heating, cooling, electromagnetic radiation,and/or light. The stimulus can be a chemical stimulus. In someinstances, a plurality of the same or different types of polymerizationapplication units can be used at different locations.

A sensor 522 can be configured to sense one or more characteristics of adroplet indicative of whether the droplet is occupied or unoccupied. Thesensor 522 may be located downstream of the junction 510. In someinstances, the sensor 522 can be an impedance sensor configured tomeasure bulk impedance when droplets pass by the sensor 522. In someinstances, a higher impedance can be measured when an occupied droplet518 passes by than when an unoccupied droplet 520 passes by. In someinstances, the sensor 522 can be an optical sensor configured to measureoptical properties of a droplet. The optical sensor and/or a supportingdevice may be configured to emit a detection signal configured to probeone or more droplets, including for example an electromagnetic signal(e.g., in any wavelength) and/or an acoustic signal. In some instances,the optical sensor and/or a supporting device may comprise anillumination source configured to illuminate the droplet or dropletswith one or more types of electromagnetic radiation. In some instances,the electromagnetic radiation can include illumination in one or more ofthe visible spectrum, infrared spectrum, the ultraviolet spectrum, andionizing radiation spectrum. In some instances, the ionizing radiationcan include x-rays. The illumination can be transillumination orepi-illumination. Alternatively the sensor 522 may be one or moredevices that are configured to provide one or more of optical sensing,thermal sensing, laser imaging, infrared imaging, capacitance sensing,mass sensing, vibration sensing across at least a portion of theelectromagnetic spectrum, and magnetic induction sensing. The sensor 522may collect sensor data on one or more properties (e.g., opticalproperties, impendence properties, etc.) or characteristics of a droplet414. For example, the one or more properties or characteristicsdetermined by the sensor 522 can be indicative of a certain type ofdroplets, such as occupied droplets, unoccupied droplets, singularlyoccupied droplets, multiply occupied droplets, droplets of a certainsize or size range, etc.

The sensor 522 can be operatively coupled to the controller 526. Forexample, the sensor 526 may transmit sensor data (e.g., one or morecharacteristics indicative of an occupancy of a droplet) to thecontroller 526. The controller 526 may then use such data, the locationof the sensor and/or the location in the fluidic channel at which theoccupied droplet 518 was detected, the time the sensor 522 detected anoccupied droplet 518, fluid flow in the channel segment 508, location ofthe light source 524, time it takes for the sensor 526 to detect and/ortransmit data to the controller 526, and/or time it takes for thecontroller 526 to send instructions to the light source 524, to sendinstructions to the light source 524 on whether or not to emit anelectromagnetic wave to polymerize a droplet. For example, using suchdata, the controller 526 may determine whether a droplet passing by thesensor 522 location at a certain time is occupied or unoccupied. Basedon the fluid flow rate in channel segment 508, the controller 526 maydetermine whether a droplet passing by the light source 524 at a certaintime (e.g., assuming time it takes for droplet to travel from the sensor522 location to the location of the light source 524 site is the sameevery time) is occupied or unoccupied.

While FIG. 5 depicts one sensor 522, it may be appreciated that theremay be a plurality of sensors, each in communication with the controller526 and/or with each other. For example, there can be a plurality ofsensors upstream or downstream of the junction 510 at differentlocations, for example, detecting the occupancy of a droplet.

While FIG. 5 depicts one controller 526 operatively coupled to both thesensor 522 and the light source 524, a separate controller can becoupled to the sensor 522 and a separate controller can be coupled tothe light source 524. The separate controllers may or may not be incommunication with each other. In some instances, there may be aplurality of controllers (e.g., two controllers to sensor 522), whereineach controller may or may not be in communication with each other. Thecontroller 526 may send instructions to sensor 522 and/or the lightsource 524 via wired connection and/or wireless connection (e.g., Wi-Fi,Bluetooth, NFC, etc.). The controller 522 may receive data from thesensor 522 and/or the light source 524 via wired connection and/orwireless connection (e.g., Wi-Fi, Bluetooth, NFC, etc.). In someinstances, the components can be directly or indirectly be incommunication with each other, with or without going through thecontroller 526. For example, the sensor 522 may be directly coupled tothe light source 526.

FIG. 6 shows an example of a microfluidic channel structure forselective polymerization of partitions based on droplet size.

The emulsion mechanism of FIG. 6 can largely parallel that of FIGS. 1,4, and 5. As shown in FIG. 6, the channel structure can include channelsegments 601, 602, 604, 606 and 608. Channel segments 601 and 602 cancommunicate at a channel junction 609. Channel segments 602, 604, 606,and 608 can communicate at a channel junction 610. In operation, a firstaqueous fluid 612 can be delivered to junction 609 from each of channelsegments 602 and 601. Cells 614 can be introduced into the junction 609via the channel segment 601 as suspensions in the first aqueous fluid612 flowing along the channel segment 601. The first aqueous fluid 612may or may not contain suspended field-attractable particles 615. Asdescribed elsewhere herein, occupied droplets and unoccupied dropletsmay be subsequently sorted via the field-attractable particles 615. Asecond fluid 616 that is immiscible with the aqueous fluid 612 isdelivered to the junction 610 from each of channel segments 604 and 606to create discrete droplets 618, 620 of the first aqueous fluid 612flowing into channel segment 608, and flowing away from junction 610. Adiscrete droplet generated may or may not include biological particles614. Each cell 614 introduced into the droplet can comprise fluorescentlabels (such as in accordance with the widely usedFluorescence-Activated Cell Sorting (FACS) mechanism).

In some instances, each droplet generated by the above emulsion may beof substantially uniform size that is appropriate (and/or acceptable)for feeding to subsequent single cell applications. In some instances,some droplets may be of substantially uniform size, and some dropletsmay have different sizes. For example, the droplets generated can have asize distribution where at least about 10%, 20%, 30%, 40%, 50%, 55%,60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or a higherpercentage of droplets generated can have a substantially uniform size.Alternatively, less than 10% of the droplets generated can have a sizedistribution where less than 10% of the droplets generated have asubstantially uniform size. The system and methods described herein mayselectively polymerize only droplets (e.g., polymerize biologicalparticles contained therein the droplets) that have the appropriate(and/or acceptable) size and/or droplets that are occupied.

The second fluid 616 can comprise an oil, such as a fluorinated oil,that includes a fluorosurfactant for stabilizing the resulting droplets,e.g., inhibiting subsequent coalescence of the resulting droplets.Examples of particularly useful partitioning fluids andfluorosurfactants are described for example, in U.S. Patent ApplicationPublication No. 2010/0105112, which is entirely incorporated herein byreference for all purposes.

The generated droplets may comprise two subsets of droplets: (1)occupied droplets 618A, 618B, containing one or more biologicalparticles 614, and (2) unoccupied droplets 620, not containing anybiological particles 614. As described above, within the subset ofoccupied droplets 618A, 618B, some droplets may have the appropriatesize (e.g., droplet 618A) and some droplets may have an inappropriatesize (e.g., droplet 618B).

A photo polymerization light source 624, such as a laser, can be locateddownstream of the junction 610 to selectively polymerize occupieddroplets. In some instances, the light source 624 can be a lamp or lightemitting diode (LED). The light source 624 can generate optical pulsesor an electromagnetic beam at a targeted direction. For example, thelight source 624 can be configured to only emit electromagnetic waveswhen an occupied droplet is passing by. The light source 624 can beoperatively coupled to a controller 626. The light source 624 canreceive instructions from the controller 626 on whether or not a dropletpassing by the light source 624, or a droplet expected to pass by thelight source at a certain time, is occupied. While FIG. 6 depicts onelight source 624, it may be appreciated that there may be a plurality oflight sources, each in communication with the controller 626 and/or witheach other. For example, a plurality of light sources may each belocated at different locations, including different upstream/downstreamlocations in the fluidic channels. Alternatively, a differentpolymerization application unit can be used to subject the droplets to astimulus, such as to trigger polymerization via heating, cooling,electromagnetic radiation, and/or light. The stimulus can be a chemicalstimulus. In some instances, a plurality of the same or different typesof polymerization application units can be used at different locations.

A sensor 622 can be configured to sense one or more characteristics of adroplet indicative of whether the droplet is occupied or unoccupied. Thesensor 622 may be located downstream of the junction 610. In someinstances, the sensor 622 can be an impedance sensor configured tomeasure bulk impedance when droplets pass by the sensor 622. In someinstances, a higher impedance can be measured when a larger droplet(e.g., droplet 618B) passes by than when a smaller droplet (e.g.,droplet 618A or droplet 620) passes by. In some instances, a higherimpedance can be measured when an occupied droplet (e.g., droplets 618A,618B) passes by than when an unoccupied droplet (e.g., droplet 620)passes by. In some instances, the sensor 622 can be an optical sensorconfigured to measure optical properties of a droplet. The opticalsensor and/or a supporting device may be configured to emit a detectionsignal configured to probe one or more droplets, including for examplean electromagnetic signal (e.g., in any wavelength) and/or an acousticsignal. In some instances, the optical sensor and/or a supporting devicemay comprise an illumination source configured to illuminate the dropletor droplets with one or more types of electromagnetic radiation. In someinstances, the electromagnetic radiation can include illumination in oneor more of the visible spectrum, infrared spectrum, the ultravioletspectrum, and ionizing radiation spectrum. In some instances, theionizing radiation can include x-rays. The illumination can betransillumination or epi-illumination. Alternatively the sensor 622 maybe one or more devices that are configured to provide one or more ofoptical sensing, thermal sensing, laser imaging, infrared imaging,capacitance sensing, mass sensing, vibration sensing across at least aportion of the electromagnetic spectrum, and magnetic induction sensing.The sensor 622 may collect sensor data on one or more properties (e.g.,optical properties, impendence properties, etc.) or characteristics of adroplet. For examples, the one or more properties and/or othercharacteristics of a droplet measured by the sensor 622 can beindicative of a size and/or an occupancy of the droplet.

The sensor 622 can be operatively coupled to the controller 626. Forexample, the sensor 626 may transmit sensor data (e.g., size and/oroccupancy of a droplet) to the controller 626. The controller 626 maythen use such data, the location of the sensor and/or the location inthe fluidic channel at which the occupancy and/or size of a droplet wasdetected, the time the sensor 622 detected, fluid flow in the channelsegment 608, location of the light source 624, time it takes for thesensor 626 to detect and/or transmit data to the controller 626, and/ortime it takes for the controller 626 to send instructions to the lightsource 624, to send instructions to the light source 624 on whether ornot to emit an electromagnetic wave to polymerize a droplet. Forexample, assuming that the fluid flow rates in the channel segment 608are substantially constant, the controller 626 may determine whether adroplet passing by a sensor 622 location at a certain time is occupiedor unoccupied. Based on the fluid flow rate in channel segment 608, thecontroller 626 may determine whether a droplet passing by the lightsource 624 at a certain time (e.g., assuming time it takes for dropletto travel from the sensor 622 location to the location of the lightsource 624 site is the same every time) is occupied or unoccupied.

While FIG. 6 depicts one sensor 622, it may be appreciated that theremay be a plurality of sensors, each in communication with the controller626 and/or with each other. For example, there can be a plurality ofsensors upstream or downstream of the junction 610 at differentlocations, for example, detecting the occupancy of a droplet. In someinstances, a separate sensor can detect occupancy of a droplet and aseparate sensor can detect size of the droplet. The separate sensors mayor may not detect such characteristics at the same upstream/downstreamlocation of the fluid channel.

While FIG. 6 depicts one controller 626 operatively coupled to both thesensor 622 and the light source 624, a separate controller can becoupled to the sensor 622 and a separate controller can be coupled tothe light source 624. The separate controllers may or may not be incommunication with each other. In some instances, there may be aplurality of controllers (e.g., two controllers to sensor 622), whereineach controller may or may not be in communication with each other. Thecontroller 626 may send instructions to sensor 622 and/or the lightsource 624 via wired connection and/or wireless connection (e.g., Wi-Fi,Bluetooth, NFC, etc.). The controller 622 may receive data from thesensor 622 and/or the light source 624 via wired connection and/orwireless connection (e.g., Wi-Fi, Bluetooth, NFC, etc.). In someinstances, the components can be directly or indirectly be incommunication with each other, with or without going through thecontroller 626. For example, the sensor 622 may be directly coupled tothe light source 626.

In some instances, a plurality of droplets, wherein a first subsetcomprises polymerized droplets and a second subset comprisespre-polymerized droplets can be further sorted based on polymerizationstatus, such as via solvent exchange. For example, the pre-polymerizeddroplets can be washed away during solvent exchange to isolate thepolymerized droplets.

The separation systems and methods for sorting and/or selectivepolymerization described above and further below may achieve superPoissonian loading. For example, the droplets can be separated into twosubsets such that at least about 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%,60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or greater of a firstsubset of droplets that is isolated are occupied droplets (e.g.,containing at least one biological particle). Such occupancy may begreater than or equal to 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%,60%, 70%, 80%, 90%, 95%, or higher. Alternatively, less than about 97%of the first subset of droplets can be occupied droplets. In someinstances, at least about 97%, 98%, 99%, or a higher percentage of asecond subset of droplets that is isolated are unoccupied droplets(e.g., not containing any biological particle and not containing anybarcode carrying beads). Alternatively, less than about 97% of thesecond subset of droplets can be unoccupied droplets. The separationsystems and methods described above and below may achieve superPoissonian monodispersity. For example, the droplets can be separatedinto two subsets such that at least about 5%, 10%, 15%, 20%, 25%, 30%,40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or greater of afirst subset of droplets that is isolated are within a given dropletsize range. Such monodispersity may be greater than or equal to 1%, 2%,3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or higher.Alternatively, less than about 97% of the first subset of droplets canbe within a given droplet size range.

As described elsewhere herein, a cell bead can be formed after thepolymerization process, such as via the selective polymerization systemsand methods described herein. Alternatively, a different polymerizationprocedure can be used. For example, cell beads can be formed byselectively polymerizing one or more biological particles withindroplets, such as via methods described in relation to FIG. 4-6. Inanother example, a cell bead can be formed by polymerizing one or morebiological particles that are suspended in a first solvent, such as oil.In some cases, a plurality of biological particles (e.g., cells) may behardened and/or polymerized in bulk by applying a stimulus (e.g., light,chemical, temperature, etc.) to the first solvent carrying the pluralityof biological particles (e.g., as suspensions). Upon formation, aplurality of cell beads may be surrounded by the first solvent, such asan oil. In order to promote integration of a cell bead into a dropletwith a gel bead, the cell bead may be placed into an aqueous environmentby a solvent exchange process. The solvent exchange process may comprisethe operations of collecting a plurality of cell beads surrounded by oil(for instance, in an Eppendorf tube or other collection vessel),removing excess oil (for instance, by pipetting), adding a ligationbuffer (such as a 3× ligation buffer), vortexing, adding a buffer (suchas a 1× 1H,1H,2H,2H-perfluoro-1-octanol (PFO) buffer), vortexing,centrifugation, and separation. The separation operation may comprisemagnetic separation.

Each of the cell beads may comprise field-attractable particles (e.g.,paramagnetic particles). For example, cell beads comprisingfield-attractable particles can be formed using the systems and methodsdescribed elsewhere herein (e.g., field-attractable particles 115 inFIG. 1 are suspended in fluids that form emulsion droplets which can bepolymerized to form cell beads). The magnetic separation may beaccomplished by using a magnetic separating apparatus to pull cell beadscontaining paramagnetic particles away from unwanted remaining oil andsolvents. In some instances, the magnetic separating apparatus can be afield application unit as described elsewhere herein (e.g., the sametype of field application unit 216 in FIG. 2A, field application unit316 in FIG. 3). For instance, the magnetic separation apparatus may beused to pull cell beads containing paramagnetic particles away from theligation buffer and PFO to allow removal of the ligation buffer and PFO(for instance by pipetting). The cell beads containing paramagneticparticles may then be suspended in a ligation buffer and vortexed. Thecell beads containing paramagnetic particles may again be separatedmagnetically and the ligation buffer may be removed. This cycle ofre-suspension, vortexing, and magnetic separation may be repeated untilthe cell beads are clean. For instance, the cycle may be repeated 1, 2,3, 4, 5, 6, 7, 8, 9, 10, or more than 10 times. The cell beads may thenbe placed into an aqueous medium.

Once the cell beads are in an aqueous medium, the cell beads may befurther treated. For instance, the cell beads in aqueous solution may befiltered (for instance, using a 70 μm filter) to remove clumps and/orlarge cell beads from the solution. In some cases, additional reagentsmay be added to and/or removed from the aqueous medium to furtherprocess the cell beads. The cell beads may then be combined intodroplets with gel beads, as described herein.

Also provided herein are the microfluidic devices used for partitioningthe cells as described above. Such microfluidic devices can comprisechannel networks for carrying out the partitioning process like thoseset forth in FIGS. 1, 4, 5, and 6. These microfluidic devices cancomprise channel networks, such as those described herein, forpartitioning cells into separate partitions, and co-partitioning suchcells with oligonucleotide barcode library members, e.g., disposed onbeads. These channel networks can be disposed within a solid body, e.g.,a glass, semiconductor or polymer body structure in which the channelsare defined, where those channels communicate at their termini withreservoirs for receiving the various input fluids, and for the ultimatedeposition of the partitioned cells, etc., from the output of thechannel networks. By way of example, and with reference to FIG. 1, areservoir fluidly coupled to channel 102 may be provided with an aqueoussuspension of cells 114, while a reservoir coupled to another channel(not shown) may be provided with an aqueous suspension of beads carryingthe oligonucleotides. Channel segments 106 and 108 may be provided witha non-aqueous solution, e.g., an oil, into which the aqueous fluids arepartitioned as droplets at the channel junction 110. Finally, an outletreservoir may be fluidly coupled to channel 108 into which thepartitioned cells and beads can be delivered and from which they may beharvested. As will be appreciated, while described as reservoirs, itwill be appreciated that the channel segments may be coupled to any of avariety of different fluid sources or receiving components, includingtubing, manifolds, or fluidic components of other systems.

Also provided are systems that control flow of these fluids through thechannel networks e.g., through applied pressure differentials,centrifugal force, electrokinetic pumping, compressors, capillary orgravity flow, or the like.

The systems and methods described herein may allow for the production ofone or more droplets containing a single biological particle and/or asingle bead. The systems and methods may also allow for the productionof one or more droplets containing a single biological particle and morethan one bead, one or more droplets containing more than one biologicalparticle and a single bead, or one or more droplets containing more thanone biological particle and more than one bead. The systems and methodsdescribed herein may allow for the production of one or more cell beadscontaining a single biological particle and/or a single bead. Thesystems and methods may also allow for selective polymerization ofoccupied droplets and/or appropriately sized droplets, which mixture ofpolymerized and pre-polymerized droplets may or may not be subjected tosubsequent sorting.

FIG. 7 shows a flowchart for a method of sorting occupied droplets andunoccupied droplets, wherein an occupied droplet contains at least abiological particle (cell) and/or a barcode carrying bead (particle).

In an operation 701, a plurality of droplets are generated upon bringinga first phase in contact with a second phase, wherein the first phaseand the second phase are immiscible. Each of the plurality of dropletscomprises some number and/or concentration of field-attractableparticles. A first subset of the plurality of droplets contains thereincells and/or barcode carrying particles, and a second subset of theplurality of droplets does not contains therein cells and/or barcodecarrying particles. A given droplet in the first subset of the pluralityof droplets may contain a single cell or a plurality of cells. A givendroplet in the first subset of the plurality of droplets may contain asingle barcode carrying particle or a plurality of barcode carryingparticles. A given droplet of the first subset of the plurality ofdroplets may contain a fewer number and/or lower concentration offield-attractable particles than a given droplet of the second subset ofthe plurality of droplets, on account of the volume occupied by the celland/or barcode carrying particles contained in the occupied droplet.

In an operation 702, the plurality of droplets is directed along thefirst channel towards an intersection of the first channel. Theintersection can be between the first channel, a second channel, and athird channel. The plurality of droplets may be directed along one ormore channels in a flow of fluid (e.g., either the first phase or thesecond phase used to generate the droplets), such as via a fluid flowunit.

In an operation 703, the plurality of droplets is subject to a forcefield, such as via a field application unit, at or near theintersection. The force field can be a magnetic field and/or an electricfield. The plurality of droplets can be subjected to the force fieldunder conditions sufficient to separate the first subset of dropletsfrom the second subset of the droplets, wherein upon separation, thefirst subset of droplets flows along the second channel, and the secondsubset of droplets flows along the third channel. For example, thefield-attractable particles can be paramagnetic particles when amagnetic field is applied. The field-attractable particles can beconductive particles when an electric field is applied. Because a givendroplet in the second subset of the plurality of droplets has a greaternumber and/or concentration of field-attractable particles than a givendroplet in the first subset of the plurality of droplets, for the samefield applied, a stronger force can act on a given droplet in the secondsubset of droplets than on a given droplet in the first subset ofdroplets. The force differential may separate the two subsets, such asby influencing a greater deviation in fluid flow path direction for agiven droplet in the second subset of droplets than for a given dropletin the first subset of droplets.

The method of FIG. 7 may isolate occupied droplets (e.g., first subsetof droplets) with super-Poissonian loading. For example, a plurality ofdroplets can be separated into two subsets such that at least about 5%,10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%,98%, 99%, or greater of a first subset of droplets that is isolated areoccupied droplets (e.g., containing at least one biological particle).Such occupancy may be greater than or equal to 1%, 2%, 3%, 4%, 5%, 10%,20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or higher. Alternatively,less than about 97% of droplets of a separated subset of the pluralityof droplets may be occupied droplets.

The method of FIG. 7 may be similarly used to isolate occupied particlesfrom a plurality of particles containing occupied and unoccupiedparticles. For example, a plurality of particles may be generated tocomprise a first subset of occupied particles (containing a biologicalparticle and/or a barcode carrying particle therein) and a second subsetof unoccupied particles, wherein particles of both the first subset andthe second subset comprise field-attractable particles. A given particleof the first subset may contain a fewer number and/or lowerconcentration of field-attractable particles than a given particle ofthe second subset, on account of the volume occupied by the cell and/orbarcode carrying particles contained in the occupied particle. Theplurality of particles may be directed along a first channel towards anintersection of the first channel. The intersection can be between thefirst channel, a second channel, and a third channel.

The plurality of particles can be subject to a force field, such as viaa field application unit, at or near the intersection. The force fieldcan be a magnetic field and/or an electric field. The plurality ofparticles can be subjected to the force field under conditionssufficient to separate the first subset from the second subset, whereinupon separation, the first subset of particles flows along the secondchannel, and the second subset of particles flows along the thirdchannel. For example, the field-attractable particles can beparamagnetic particles when a magnetic field is applied. Thefield-attractable particles can be conductive particles when an electricfield is applied. Because a given particle in the second subset has agreater number and/or concentration of field-attractable particles thana given particle in the first subset, for the same field applied, astronger force can act on a given particle in the second subset than ona given particle in the first subset. The force differential mayseparate the two subsets, such as by influencing a greater deviation influid flow path direction for a given particle in the second subset thanfor a given particle in the first subset.

The method of FIG. 7 may isolate occupied particles (e.g., first subsetof particles) with super-Poissonian loading. For example, a plurality ofparticles can be separated into two subsets such that at least about 5%,10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%,98%, 99%, or greater of a first subset of particles that is isolated areoccupied particles (e.g., containing at least one biological particle).Such occupancy may be greater than or equal to 1%, 2%, 3%, 4%, 5%, 10%,20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or higher. Alternatively,less than about 97% of particles of a separated subset of the pluralityof particles may be occupied particles.

FIG. 8 shows a flowchart for a method of sorting occupied droplets andunoccupied droplets, wherein an occupied droplet contains at least abiological particle (cell) and/or a barcode carrying bead (particle).

In an operation 801, a plurality of droplets are generated upon bringinga first phase in contact with a second phase, wherein the first phaseand the second phase are immiscible. Each of the plurality of dropletscomprises may or may not comprise some number and/or concentration offield-attractable particles. The presence of field-attractable particlesis not required. A first subset of the plurality of droplets containstherein cells and/or barcode carrying particles, and a second subset ofthe plurality of droplets does not contain therein cells and/or barcodecarrying particles. A given droplet in the first subset of the pluralityof droplets may contain a single cell or a plurality of cells. A givendroplet in the first subset of the plurality of droplets may contain asingle barcode carrying particle or a plurality of barcode carryingparticles.

In an operation 802, the plurality of droplets is directed along thefirst channel towards an intersection of the first channel. Theintersection can be between the first channel, a second channel, and athird channel. The plurality of droplets may be directed along one ormore channels in a flow of fluid (e.g., either the first phase or thesecond phase used to generate the droplets), such as via a fluid flowunit.

In an operation 803, the plurality of droplets is subject to a pressurepulse, such as via a pressure application unit, at or near theintersection. The pressure pulse can be provided as a positive pressurepulse or a negative pressure pulse. The plurality of droplets can besubjected to the pressure pulse under conditions sufficient to separatethe first subset of droplets from the second subset of the droplets,wherein upon separation, the first subset of droplets flows along thesecond channel, and the second subset of droplets flows along the thirdchannel. Because a given droplet in the second subset of the pluralityof droplets has different particle and/or suspension characteristics inthe fluid than a given droplet in the first subset of the plurality ofdroplets, for the same pressure pulse applied, a different hydrodynamicforce can act on a given droplet in the second subset of droplets thanon a given droplet in the first subset of droplets. The pressure pulsemay separate the two subsets, such as by influencing a greater deviationin fluid flow path direction for a given droplet in the second subset ofdroplets than for a given droplet in the first subset of droplets.

In some instances, a sensor, such as an impedance sensor or an opticalsensor, may measure one or more characteristics of a droplet at anupstream location of the intersection. The sensing data may beindicative of the occupancy of the droplet. The sensing data may betransmitted to a controller. The controller may use the sensing data todetermine the occupancy of the droplet and instruct the pressureapplication unit to generate or not generate pressure pulses to separatethe first subset from the second subset.

The method of FIG. 8 may isolate occupied droplets (e.g., first subsetof droplets) with super-Poissonian loading. For example, a plurality ofdroplets can be separated into two subsets such that at least about 5%,10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%,98%, 99%, or greater of a first subset of droplets that is isolated areoccupied droplets (e.g., containing at least one biological particle).Such occupancy may be greater than or equal to 1%, 2%, 3%, 4%, 5%, 10%,20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or higher. Alternatively,less than about 97% of droplets of a separated subset of the pluralityof droplets may be occupied droplets.

The method of FIG. 8 may be similarly used to isolate cell beads from aplurality of particles containing both cell beads and unoccupiedparticles. For example, a plurality of particles may be generated tocomprise a first subset of occupied particles (containing a biologicalparticle and/or a barcode carrying particle therein) and a second subsetof unoccupied particles. The presence of field-attractable particles isnot required. The plurality of particles may be directed along a firstchannel towards an intersection of the first channel. The intersectioncan be between the first channel, a second channel, and a third channel.The plurality of particles may be subject to a pressure pulse, such asvia a pressure application unit, at or near the intersection. Thepressure pulse can be provided as a positive pressure pulse or anegative pressure pulse.

The plurality of particles can be subjected to the pressure pulse underconditions sufficient to separate the first subset of particles (e.g.,cell beads) from the second subset of the particles (e.g., unoccupiedparticles), wherein upon separation, the first subset of particles flowsalong the second channel, and the second subset of particles flows alongthe third channel. Because a given particle in the second subset of theplurality of particles has different particle and/or suspensioncharacteristics in the fluid than a given particle in the first subsetof the plurality of particles, for the same pressure pulse applied, adifferent hydrodynamic force can act on a given particle in the secondsubset of particles than on a given particle in the first subset ofparticles. The pressure pulse may separate the two subsets, such as byinfluencing a greater deviation in fluid flow path direction for a givenparticle in the second subset of particles than for a given particle inthe first subset of particles.

In some instances, a sensor, such as an impedance sensor or an opticalsensor, may measure one or more characteristics of a particle at anupstream location of the intersection. The sensing data may beindicative of the occupancy of the particle. The sensing data may betransmitted to a controller. The controller may use the sensing data todetermine the occupancy of the particle and instruct the pressureapplication unit to generate or not generate pressure pulses to separatethe first subset from the second subset.

The method of FIG. 8 may isolate cell beads (e.g., first subset ofparticles) with super-Poissonian loading. For example, a plurality ofparticles can be separated into two subsets such that at least about 5%,10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%,98%, 99%, or greater of a first subset of particles that is isolated arecell beads (e.g., containing at least one biological particle). Suchoccupancy may be greater than or equal to 1%, 2%, 3%, 4%, 5%, 10%, 20%,30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or higher. Alternatively, lessthan about 97% of particles of a separated subset of the plurality ofparticles may be cell beads.

FIG. 9 shows a flowchart for a method of selectively polymerizingoccupied droplets, wherein an occupied droplet contains at least abiological particle (cell) and/or a barcode carrying bead (particle).

In an operation 901, a plurality of droplets are generated upon bringinga first phase in contact with a second phase, wherein the first phaseand the second phase are immiscible. Each of the plurality of dropletscomprises may or may not comprise some number and/or concentration offield-attractable particles. The presence of field-attractable particlesis not required. A first subset of the plurality of droplets containstherein cells and/or barcode carrying particles, and a second subset ofthe plurality of droplets does not contain therein cells and/or barcodecarrying particles. A given droplet in the first subset of the pluralityof droplets may contain a single cell or a plurality of cells. A givendroplet in the first subset of the plurality of droplets may contain asingle barcode carrying particle or a plurality of barcode carryingparticles.

In an operation 902, at an upstream location, a sensor detects and/ormeasures one or more characteristics of a droplet passing through theupstream location. The one or more characteristics can be indicative ofthe occupancy of the droplet. In some instances, the sensor can be animpedance sensor configured to detect bulk impedance as a droplet or aplurality of droplets passes through the upstream location. A higherimpedance can be measured for occupied droplets than for unoccupieddroplets. In some instances, the sensor can be an optical sensorconfigured to detect one or more optical characteristics of the dropletas the droplet passes through the upstream location. Alternatively, aplurality of the same or different types of sensors can be used todetect and/or measure one or more characteristics of the droplet passingthrough the upstream location.

In an operation 903, the sensing data may transmitted to a controller.For example, the sensor can be operatively coupled to the controller.The controller can determine, based at least in part on the sensor data,whether a droplet passing through a downstream location is occupied orunoccupied. For example, the controller may use such sensor data, thelocation of the sensor and/or the location in the fluidic channel atwhich the occupancy of a droplet was detected, the time the sensordetected the occupancy of a droplet, fluid flow rate of one or morechannels, location of the light source, time it takes for the sensor todetect and/or transmit data to the controller, and/or time it takes forthe controller to send instructions to the light source, to sendinstructions to the light source on whether or not to emit anelectromagnetic wave to polymerize a droplet.

Alternatively, in some instances, the sensor may detect the presence ofa cell suspended in a fluid flow before the droplets are generated(e.g., at the intersection), and use such sensor data of the presence ofthe cell, and other information (e.g., location, times, fluid flowrates) to determine whether a droplet at a downstream location isoccupied.

In an operation 904, the controller may transmit instruction to a lightsource at a downstream location to emit electromagnetic waves topolymerize a droplet if the droplet is occupied, and not to emitelectromagnetic waves if the droplet is unoccupied, thus letting theunoccupied droplet pass through unpolymerized. Alternatively, otherpolymerization application units can be used in place of, or inconjunction with the light source.

The method of FIG. 9 may polymerize occupied droplets (e.g., firstsubset of droplets) with super-Poissonian distribution. The separationsystems and methods disclosed herein may achieve super Poisson loading.For example, at least about 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%,70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or greater of a plurality ofdroplets that are polymerized can be occupied droplets. Such occupancymay be greater than or equal to 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%,50%, 60%, 70%, 80%, 90%, 95%, or higher. Alternatively, less than about97% of polymerized droplets may be occupied droplets.

FIG. 10 shows a flowchart for a method of selectively polymerizingappropriately sized droplets.

In an operation 1001, a plurality of droplets are generated uponbringing a first phase in contact with a second phase, wherein the firstphase and the second phase are immiscible. Each of the plurality ofdroplets comprises may or may not comprise some number and/orconcentration of field-attractable particles. The presence offield-attractable particles is not required. A first subset of theplurality of droplets contains therein cells and/or barcode carryingparticles, and a second subset of the plurality of droplets does notcontain therein cells and/or barcode carrying particles. A given dropletin the first subset of the plurality of droplets may contain a singlecell or a plurality of cells. A given droplet in the first subset of theplurality of droplets may contain a single barcode carrying particle ora plurality of barcode carrying particles.

In an operation 1002, at an upstream location, a sensor detects and/ormeasures one or more characteristics of a droplet passing through theupstream location. The one or more characteristics can be indicative ofa size of the droplet. In some instances, the sensor can be an impedancesensor configured to detect bulk impedance as a droplet or a pluralityof droplets passes through the upstream location. A higher impedance canbe measured for larger droplets than for smaller droplets. In someinstances, the sensor can be an optical sensor configured to detect oneor more optical characteristics of the droplet as the droplet passesthrough the upstream location. Alternatively, a plurality of the same ordifferent types of sensors can be used to detect and/or measure one ormore characteristics of the droplet passing through the upstreamlocation.

In an operation 1003, the sensing data may transmitted to a controller.For example, the sensor can be operatively coupled to the controller.The controller can determine, based at least in part on the sensor data,whether a droplet passing through a downstream location is appropriatelysized or inappropriately sized. For example, the controller may use suchsensor data, the location of the sensor and/or the location in thefluidic channel at which the size of a droplet was detected, the timethe sensor detected the size of a droplet, fluid flow rate of one ormore channels, location of the light source, time it takes for thesensor to detect and/or transmit data to the controller, and/or time ittakes for the controller to send instructions to the light source, tosend instructions to the light source on whether or not to emit anelectromagnetic wave to polymerize a droplet.

In an operation 1004, the controller may transmit instruction to a lightsource at a downstream location to emit electromagnetic waves topolymerize a droplet if the droplet is appropriately sized, and not toemit electromagnetic waves if the droplet is inappropriately sized, thusletting the inappropriately sized droplet pass through unpolymerized.Alternatively, other polymerization application units can be used inplace of, or in conjunction with the light source.

The method of FIG. 10 may polymerize appropriately sized droplets (e.g.,first subset of droplets) with super-Poissonian distribution. Forexample, at least about 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%,80%, 90%, 95%, 96%, 97%, 98%, 99%, or greater of a plurality of dropletsthat is polymerized can be appropriately sized droplets. Alternatively,less than about 97% of polymerized droplets may be appropriately sizeddroplets.

In some instances, the methods of FIG. 9 and FIG. 10 can be combinedsuch that one or more sensors detect one or more characteristics of adroplet at an upstream location (or upstream locations), wherein the oneor more characteristics of the droplet are indicative of both a size andan occupancy of the droplet, and transmits the sensor data to acontroller. The controller transmits instructions to a light source at adownstream location to polymerize the droplet only if the droplet isboth appropriately sized and occupied.

In another aspect, provided is a passive mechanism for sorting occupieddroplets from unoccupied droplets. The passive mechanism may not requireapplication of external forces (e.g., magnetic field, electric field,pressure pulse, etc.) on the droplets to achieve sorting. The passivemechanism may sort droplets based at least in part on mechanicalproperties of the droplets. For example, the passive mechanism may sortdroplets based at least in part on properties such as deformability andsurface tension (e.g., surface interface energy) of the droplets. Insome instances, due to the presence of one or more biological particlesin an occupied droplet, the occupied droplet may demonstrate lowerdeformability and/or higher surface tension properties than unoccupieddroplets, making occupied droplets ‘harder’ or ‘stiffer’ than unoccupieddroplets. Thus, when a plurality of droplets comprising both a firstsubset of occupied droplets and a second subset of unoccupied dropletsis directed to pass through an aperture which is smaller in size than adiameter of a given droplet in the plurality of droplets, only thosedroplets capable of deforming (e.g., unoccupied droplets having higherdeformability properties) may pass through the aperture, trapping theoccupied droplets, thereby sorting the occupied droplets from theunoccupied droplets.

FIG. 11 shows a schematic example of a microfluidic channel structurefor separating occupied droplets from unoccupied droplets. As describedelsewhere herein, when droplets are generated, there may be at least afirst subset population of occupied droplets containing one or morebiological particles and at least a second subset population ofunoccupied droplets not containing any biological particles. In somecases, the droplets may additionally contain one or more barcodecarrying beads. For example, a droplet may have only a biologicalparticle, a droplet may have only a barcode carrying bead, a droplet mayhave both a biological particle and a barcode carrying bead, or adroplet may have neither biological particles nor barcode carryingbeads. In some cases, the majority of occupied partitions (e.g.,droplets) can include no more than one biological particle per occupiedpartition and, in some cases, some of the generated partitions can beunoccupied (e.g., by any biological particle). In some cases, though,some of the occupied partitions may include more than one biologicalparticle. In some cases, the partitioning process may be controlled suchthat fewer than 25% of the occupied partitions contain more than onebiological particle, fewer than 20% of the occupied partitions have morethan one biological particle, or fewer than 10% or even fewer than 5% ofthe occupied partitions include more than one biological particle perpartition.

As shown in FIG. 11, the channel structure can include a channel segment1100 with an entrance 1102 and exit 1104. In some instances, the outflowchannel 108 of the emulsion carrying the generated droplets in FIG. 1can be upstream of the channel segment 1100. A fluid flow unit (notshown) can be configured to facilitate flow of fluid in the channelstructure.

In operation, a plurality of discrete droplets, each comprising a firstaqueous fluid 1100 can flow as emulsions in a second fluid 1112, whereinthe second fluid 1112 is immiscible to the first aqueous fluid 1110. Thedroplets being transported along channel segment 1100 can comprise afirst subset of droplets 1108 that are each occupied with at least abiological particle and/or a barcode carrying bead and a second subsetof droplets 1110 that are each unoccupied. As described above, a givenunoccupied droplet can have a higher deformability and/or lower surfacetension property than a given occupied droplet, due to the presence ofone or more biological particles in the occupied droplet.

The channel segment 1100 can comprise a plurality of entrapmentstructures 1114. An entrapment structure can define an aperture. A sizeof the aperture may be less than a diameter (or other size dimension) ofa droplet. A size of the aperture may be less than a minimum dimensionof a droplet. In some instances, the size of the aperture can be at mostabout 90%, 80%, 70%, 60%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%,or 5% of the diameter of a droplet (in a pre-deformed state). The sizeof the aperture can be less than about 5% of the diameter of a droplet.Alternatively, the size of the aperture can be greater than 50%, 60%,70%, 80%, 90%, 100%, 150%, or 200% of the diameter of a droplet. Thesize of the aperture can be greater than the diameter of the droplet.When a plurality of droplets comprising both a first subset of occupieddroplets 1108 and a second subset of unoccupied droplets 1110 isdirected to pass through entrapment structures 1114 defining apertureswhich is smaller in size than a diameter of a given droplet in theplurality of droplets, only those droplets deforming 1118 (e.g.,unoccupied droplets 1110 having higher deformability properties) suchthat at least one dimension of the droplet is less than a size of theaperture may pass through one or more apertures defined by theentrapment structures 1114. Because occupied droplets 1108 may be‘harder’ or ‘stiffer,’ due to a presence of one or more biologicalparticles in the droplets, the occupied droplets may resist deformation,at least from deforming to a size smaller than a size of the aperture,and remain trapped by the entrapment structures 1114.

In some embodiments, there may exist more entrapment structures 1114(and thus more apertures) in the channel structure than there aredroplets 1106, 1108 passing through the channel structure. Beneficially,when the occupied droplets 1108 are prevented from flowing through theentrapment structures 1114, and the occupied droplets 1108 clog (orblock) some apertures of the entrapment structures 1114, the unoccupieddroplets 1110 may still deform and flow through other apertures. Aftersorting, the entrapment structures 1114 may retain from the plurality ofdroplets only the first subset of occupied droplets 1108. The unoccupieddroplets 1104 may flow through all entrapment structures 1114 and exitthe channel segment 1100 to a separate compartment, such as forrecycling or discarding. While FIG. 11 shows exemplary configurationsand a layout of entrapment structures in the channel structure, theconfigurations and layout of entrapment structures are not limited assuch. For example, an entrapment structure can be a single plate with aplurality of apertures (e.g., holes) defined in the plate. The plate canbe planar, curved, and/or a combination thereof. The channel structureand the entrapment structures 1114 can be configured such that a dropletfrom the plurality of droplets passes through at least one entrapmentstructure (and aperture defined therein). After entrapment and/orsorting of the occupied droplets, the fluid flow unit (not shown) can beconfigured to reverse a fluid flow direction to collect the occupieddroplets from the entrapment structures.

In some instances, the fluid flow unit may comprise a compressor toprovide positive pressure at an upstream location to direct the fluidfrom the upstream location to flow to a downstream location. In someinstances, the fluid flow unit may comprise a pump to provide negativepressure at a downstream location to direct the fluid from an upstreamlocation to flow to the downstream location. In some instances, thefluid flow unit may comprise both a compressor and a pump, each atdifferent locations. In some instances, the fluid flow unit may comprisedifferent devices at different locations. The fluid flow unit maycomprise an actuator.

The systems and methods described with respect to FIG. 11 may be used toseparate occupied particles (e.g., cell beads) from unoccupiedparticles. As described elsewhere herein, a plurality of particles maycomprise a first subset of particles occupied by biological particles(e.g., cells) and a second subset of particles unoccupied by biologicalparticles. As described above, a given unoccupied particle can have ahigher deformability and/or lower surface tension property than a givenoccupied particle (e.g., cell bead) due to the presence of one or morebiological particles in the occupied particle. In a channel structureincluding channel segment 1100 with an entrance 1102 and exit 1104, andcomprising the plurality of entrapment structures 1114, the plurality ofparticles may be directed to flow (e.g., as suspensions in a fluid,e.g., aqueous fluid) along the channel segment 1100 from entrance 1102to exit 1104 across the plurality of entrapment structures 1114.

An entrapment structure can define an aperture. A size of the aperturemay be less than a diameter (or other size dimension) of a particle. Asize of the aperture may be less than a minimum dimension of a particle.In some instances, the size of the aperture can be at most about 90%,80%, 70%, 60%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, or 5% of thediameter of a particle (in a pre-deformed state). The size of theaperture can be less than about 5% of the diameter of a particle.Alternatively, the size of the aperture can be greater than 50%, 60%,70%, 80%, 90%, 100%, 150%, or 200% of the diameter of a particle. Thesize of the aperture can be greater than the diameter of the particle.When a plurality of particles comprising both a first subset of occupiedparticles 1108 and a second subset of unoccupied particles 1110 isdirected to pass through entrapment structures 1114 defining apertureswhich is smaller in size than a diameter of a given particle in theplurality of particles, only those particles deforming 1118 (e.g.,unoccupied particles 1110 having higher deformability properties) suchthat at least one dimension of the particle is less than a size of theaperture may pass through one or more apertures defined by theentrapment structures 1114. Because occupied particles 1108 (e.g., cellbeads) may be harder or stiffer, due to a presence of one or morebiological particles in the particles, the occupied particles may resistdeformation, at least from deforming to a size smaller than a size ofthe aperture, and remain trapped by the entrapment structures 1114.

In some embodiments, there may exist more entrapment structures 1114(and thus more apertures) in the channel structure than there areparticles 1106, 1108 passing through the channel structure.Beneficially, when the occupied particles 1108 are prevented fromflowing through the entrapment structures 1114, and the occupiedparticles 1108 clog (or block) some apertures of the entrapmentstructures 1114, the unoccupied particles 1110 may still deform and flowthrough other apertures. After sorting, the entrapment structures 1114may retain from the plurality of particles only the first subset ofoccupied particles 1108. The unoccupied particles 1104 may flow throughall entrapment structures 1114 and exit the channel segment 1100 to aseparate compartment, such as for recycling or discarding.

The separation systems and methods disclosed herein (such as withreference to FIG. 11) may achieve super Poisson loading. For example,the droplets can be separated into two subsets such that at least about5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%,97%, 98%, 99%, or greater of a first subset of droplets that is isolatedare occupied droplets (e.g., containing at least one biologicalparticle). Such occupancy may be greater than or equal to 1%, 2%, 3%,4%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or higher.Alternatively, less than about 97% of the first subset of droplets canbe occupied droplets. In some instances, at least about 97%, 98%, 99%,or a higher percentage of a second subset of droplets that is isolatedcan be unoccupied droplets (e.g., not containing any biological particleand not containing any barcode carrying beads). Alternatively, less thanabout 97% of the second subset of droplets can be unoccupied droplets.For example, the plurality of particles can be separated into twosubsets such that at least about 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%,60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or greater of a firstsubset of particles that is isolated are cell beads (e.g., containing atleast one biological particle). Such occupancy may be greater than orequal to 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%,90%, 95%, or higher. Alternatively, less than about 97% of the firstsubset of particles can be cell beads. In some instances, at least about97%, 98%, 99%, or a higher percentage of a second subset of particlesthat is isolated can be unoccupied particles (e.g., not containing anybiological particle and not containing any barcode carrying beads).Alternatively, less than about 97% of the second subset of particles canbe unoccupied particles.

Microfluidic Architectures

In an aspect, provided herein are various microfluidic architecturesthat can be used in conjunction with the systems and methods describedherein.

In accordance with certain aspects, beads may be delivered to droplets.FIG. 12 shows an example of a microfluidic channel structure 1200 fordelivering barcode carrying beads to droplets. An example of a barcodecarrying bead is described with respect to FIG. 19. The channelstructure 1200 can include channel segments 1201, 1202, 1204, 1206 and1208 communicating at a channel junction 1210. In operation, the channelsegment 1201 may transport an aqueous fluid 1212 that includes aplurality of beads 1214 (e.g., with nucleic acid molecules,oligonucleotides, molecular tags) along the channel segment 1201 intojunction 1210. The plurality of beads 1214 may be sourced from asuspension of beads. For example, the channel segment 1201 may beconnected to a reservoir comprising an aqueous suspension of beads 1214.The channel segment 1202 may transport the aqueous fluid 1212 thatincludes a plurality of biological particles 1216 along the channelsegment 1202 into junction 1210. The plurality of biological particles1216 may be sourced from a suspension of biological particles. Forexample, the channel segment 1202 may be connected to a reservoircomprising an aqueous suspension of biological particles 1216. In someinstances, the aqueous fluid 1212 in either the first channel segment1201 or the second channel segment 1202, or in both segments, caninclude one or more reagents, as further described below. A second fluid1218 that is immiscible with the aqueous fluid 1212 (e.g., oil) can bedelivered to the junction 1210 from each of channel segments 1204 and1206. Upon meeting of the aqueous fluid 1212 from each of channelsegments 1201 and 1202 and the second fluid 1218 from each of channelsegments 1204 and 1206 at the channel junction 1210, the aqueous fluid1212 can be partitioned as discrete droplets 1220 in the second fluid1218 and flow away from the junction 1210 along channel segment 1208.The channel segment 1208 may deliver the discrete droplets to an outletreservoir fluidly coupled to the channel segment 1208, where they may beharvested.

As an alternative, the channel segments 1201 and 1202 may meet atanother junction upstream of the junction 1210. At such junction, beadsand biological particles may form a mixture that is directed alonganother channel to the junction 1210 to yield droplets 1220. The mixturemay provide the beads and biological particles in an alternatingfashion, such that, for example, a droplet comprises a single bead and asingle biological particle.

Beads, biological particles and droplets may flow along channels atsubstantially regular flow profiles (e.g., at regular flow rates). Suchregular flow profiles may permit a droplet to include a single bead anda single biological particle. Such regular flow profiles may permit thedroplets to have an occupancy (e.g., droplets having beads andbiological particles) greater than 5%, 10%, 20%, 30%, 40%, 50%, 60%,70%, 80%, 90%, or 95%. Such regular flow profiles and devices that maybe used to provide such regular flow profiles are provided in, forexample, U.S. Patent Publication No. 12015/0292988, which is entirelyincorporated herein by reference.

The second fluid 1218 can comprise an oil, such as a fluorinated oil,that includes a fluorosurfactant for stabilizing the resulting droplets,for example, inhibiting subsequent coalescence of the resulting droplets1220.

A discrete droplet that is generated may include an individualbiological particle 1216. A discrete droplet that is generated mayinclude a barcode or other reagent carrying bead 1214. A discretedroplet generated may include both an individual biological particle anda barcode carrying bead, such as droplets 1220. In some instances, adiscrete droplet may include more than one individual biologicalparticle or no biological particle. In some instances, a discretedroplet may include more than one bead or no bead. A discrete dropletmay be unoccupied (e.g., no beads, no biological particles).

Beneficially, a discrete droplet partitioning a biological particle anda barcode carrying bead may effectively allow the attribution of thebarcode to macromolecular constituents of the biological particle withinthe partition. The contents of a partition may remain discrete from thecontents of other partitions.

As will be appreciated, the channel segments described herein may becoupled to any of a variety of different fluid sources or receivingcomponents, including reservoirs, tubing, manifolds, or fluidiccomponents of other systems. As will be appreciated, the microfluidicchannel structure 1200 may have other geometries. For example, amicrofluidic channel structure can have more than one channel junctions.For example, a microfluidic channel structure can have 2, 3, 4, or 5channel segments each carrying beads that meet at a channel junction.Fluid may be directed flow along one or more channels or reservoirs viaone or more fluid flow units. A fluid flow unit can comprise compressors(e.g., providing positive pressure), pumps (e.g., providing negativepressure), actuators, and the like to control flow of the fluid. Fluidmay also or otherwise be controlled via applied pressure differentials,centrifugal force, electrokinetic pumping, vacuum, capillary or gravityflow, or the like.

In accordance with certain aspects, biological particles may bepartitioned along with lysis reagents in order to release the contentsof the biological particles within the partition. In such cases, thelysis agents can be contacted with the biological particle suspensionconcurrently with, or immediately prior to, the introduction of thebiological particles into the partitioning junction/droplet generationzone, such as through an additional channel or channels upstream of thechannel junction. In accordance with other aspects, additionally oralternatively, biological particles may be partitioned along with otherreagents, as will be described further below.

FIG. 13 shows an example of a microfluidic channel structure 1300 forco-partitioning biological particles and reagents. The channel structure1300 can include channel segments 1301, 1302, 1304, 1306 and 1308.Channel segments 1301 and 1302 communicate at a first channel junction1309. Channel segments 1302, 1304, 1306, and 1308 communicate at asecond channel junction 1310.

In an example operation, the channel segment 1301 may transport anaqueous fluid 1312 that includes a plurality of biological particles1314 along the channel segment 1301 into the second junction 1310. As analternative or in addition to, channel segment 1301 may transport beads(e.g., gel beads). The beads may comprise barcode molecules.

For example, the channel segment 1301 may be connected to a reservoircomprising an aqueous suspension of biological particles 1314. Upstreamof, and immediately prior to reaching, the second junction 1310, thechannel segment 1301 may meet the channel segment 1302 at the firstjunction 1309. The channel segment 1302 may transport a plurality ofreagents 1315 (e.g., lysis agents) suspended in the aqueous fluid 1312along the channel segment 1302 into the first junction 1309. Forexample, the channel segment 1302 may be connected to a reservoircomprising the reagents 1315. After the first junction 1309, the aqueousfluid 1312 in the channel segment 1301 can carry both the biologicalparticles 1314 and the reagents 1315 towards the second junction 1310.In some instances, the aqueous fluid 1312 in the channel segment 1301can include one or more reagents, which can be the same or differentreagents as the reagents 1315. A second fluid 1316 that is immisciblewith the aqueous fluid 1312 (e.g., oil) can be delivered to the secondjunction 1310 from each of channel segments 1304 and 1306. Upon meetingof the aqueous fluid 1312 from the channel segment 1301 and the secondfluid 1316 from each of channel segments 1304 and 1306 at the secondchannel junction 1310, the aqueous fluid 1312 can be partitioned asdiscrete droplets 1318 in the second fluid 1316 and flow away from thesecond junction 1310 along channel segment 1308. The channel segment1308 may deliver the discrete droplets 1318 to an outlet reservoirfluidly coupled to the channel segment 1308, where they may beharvested.

The second fluid 1316 can comprise an oil, such as a fluorinated oil,that includes a fluorosurfactant for stabilizing the resulting droplets,for example, inhibiting subsequent coalescence of the resulting droplets1318.

A discrete droplet generated may include an individual biologicalparticle 1314 and/or one or more reagents 1315. In some instances, adiscrete droplet generated may include a barcode carrying bead (notshown), such as via other microfluidics structures described elsewhereherein. In some instances, a discrete droplet may be unoccupied (e.g.,no reagents, no biological particles).

Beneficially, when lysis reagents and biological particles areco-partitioned, the lysis reagents can facilitate the release of thecontents of the biological particles within the partition. The contentsreleased in a partition may remain discrete from the contents of otherpartitions.

As will be appreciated, the channel segments described herein may becoupled to any of a variety of different fluid sources or receivingcomponents, including reservoirs, tubing, manifolds, or fluidiccomponents of other systems. As will be appreciated, the microfluidicchannel structure 1300 may have other geometries. For example, amicrofluidic channel structure can have more than two channel junctions.For example, a microfluidic channel structure can have 2, 3, 4, 5channel segments or more each carrying the same or different types ofbeads, reagents, and/or biological particles that meet at a channeljunction. Fluid flow in each channel segment may be controlled tocontrol the partitioning of the different elements into droplets. Fluidmay be directed flow along one or more channels or reservoirs via one ormore fluid flow units. A fluid flow unit can comprise compressors (e.g.,providing positive pressure), pumps (e.g., providing negative pressure),actuators, and the like to control flow of the fluid. Fluid may also orotherwise be controlled via applied pressure differentials, centrifugalforce, electrokinetic pumping, vacuum, capillary or gravity flow, or thelike.

In some aspects, provided are systems and methods for controlledpartitioning. Droplet size may be controlled by adjusting certaingeometric features in channel architecture (e.g., microfluidics channelarchitecture). For example, an expansion angle, width, and/or length ofa channel may be adjusted to control droplet size.

FIG. 14 shows an example of a microfluidic channel structure for thecontrolled partitioning of beads into discrete droplets. A channelstructure 1400 can include a channel segment 1402 communicating at achannel junction 1406 (or intersection) with a reservoir 1404. Thereservoir 1404 can be a chamber. Any reference to “reservoir,” as usedherein, can also refer to a “chamber.” In operation, an aqueous fluid1408 that includes suspended beads 1412 may be transported along thechannel segment 1402 into the junction 1406 to meet a second fluid 1410that is immiscible with the aqueous fluid 1408 in the reservoir 1404 tocreate droplets 1416, 1418 of the aqueous fluid 1408 flowing into thereservoir 1404. At the junction 1406 where the aqueous fluid 1408 andthe second fluid 1410 meet, droplets can form based on factors such asthe hydrodynamic forces at the junction 1406, flow rates of the twofluids 1408, 1410, fluid properties, and certain geometric parameters(e.g., w, h₀, α, etc.) of the channel structure 1400. A plurality ofdroplets can be collected in the reservoir 1404 by continuouslyinjecting the aqueous fluid 1408 from the channel segment 1402 throughthe junction 1406.

A discrete droplet generated may include a bead (e.g., as in occupieddroplets 1416). Alternatively, a discrete droplet generated may includemore than one bead. Alternatively, a discrete droplet generated may notinclude any beads (e.g., as in unoccupied droplet 1418). In someinstances, a discrete droplet generated may contain one or morebiological particles, as described elsewhere herein. In some instances,a discrete droplet generated may comprise one or more reagents, asdescribed elsewhere herein.

In some instances, the aqueous fluid 1408 can have a substantiallyuniform concentration or frequency of beads 1412. The beads 1412 can beintroduced into the channel segment 1402 from a separate channel (notshown in FIG. 14). The frequency of beads 1412 in the channel segment1402 may be controlled by controlling the frequency in which the beads1412 are introduced into the channel segment 1402 and/or the relativeflow rates of the fluids in the channel segment 1402 and the separatechannel. In some instances, the beads can be introduced into the channelsegment 1402 from a plurality of different channels, and the frequencycontrolled accordingly.

In some instances, the aqueous fluid 1408 in the channel segment 1402can comprise biological particles. In some instances, the aqueous fluid1408 can have a substantially uniform concentration or frequency ofbiological particles. As with the beads, the biological particles can beintroduced into the channel segment 1402 from a separate channel. Thefrequency or concentration of the biological particles in the aqueousfluid 1408 in the channel segment 1402 may be controlled by controllingthe frequency in which the biological particles are introduced into thechannel segment 1402 and/or the relative flow rates of the fluids in thechannel segment 1402 and the separate channel. In some instances, thebiological particles can be introduced into the channel segment 1402from a plurality of different channels, and the frequency controlledaccordingly. In some instances, a first separate channel can introducebeads and a second separate channel can introduce biological particlesinto the channel segment 1402. The first separate channel introducingthe beads may be upstream or downstream of the second separate channelintroducing the biological particles.

The second fluid 1410 can comprise an oil, such as a fluorinated oil,that includes a fluorosurfactant for stabilizing the resulting droplets,for example, inhibiting subsequent coalescence of the resultingdroplets.

In some instances, the second fluid 1410 may not be subjected to and/ordirected to any flow in or out of the reservoir 1404. For example, thesecond fluid 1410 may be substantially stationary in the reservoir 1404.In some instances, the second fluid 1410 may be subjected to flow withinthe reservoir 1404, but not in or out of the reservoir 1404, such as viaapplication of pressure to the reservoir 1404 and/or as affected by theincoming flow of the aqueous fluid 1408 at the junction 1406.Alternatively, the second fluid 1410 may be subjected and/or directed toflow in or out of the reservoir 1404. For example, the reservoir 1404can be a channel directing the second fluid 1410 from upstream todownstream, transporting the generated droplets.

The channel structure 1400 at or near the junction 1406 may have certaingeometric features that at least partly determine the sizes of thedroplets formed by the channel structure 1400. The channel segment 1402can have a height, h₀ and width, w, at or near the junction 1406. By wayof example, the channel segment 1402 can comprise a rectangularcross-section that leads to a reservoir 1404 having a widercross-section (such as in width or diameter). Alternatively, thecross-section of the channel segment 1402 can be other shapes, such as acircular shape, trapezoidal shape, polygonal shape, or any other shapes.The top and bottom walls of the reservoir 1404 at or near the junction1406 can be inclined at an expansion angle, α. The expansion angle, α,allows the tongue (portion of the aqueous fluid 1408 leaving channelsegment 1402 at junction 1406 and entering the reservoir 1404 beforedroplet formation) to increase in depth and facilitate decrease incurvature of the intermediately formed droplet. Droplet size maydecrease with increasing expansion angle. The resulting droplet radius,R_(d), may be predicted by the following equation for the aforementionedgeometric parameters of h₀, w, and α:

$R_{d} \approx {0.44\left( {1 + {2.2\sqrt{\tan\mspace{14mu}\alpha}\frac{w}{h_{0}}}} \right)\frac{h_{0}}{\sqrt{\tan\mspace{14mu}\alpha}}}$

By way of example, for a channel structure with w=21 μm, h=21 μm, andα=3°, the predicted droplet size is 121 μm. In another example, for achannel structure with w=25 h=25 μm, and α=5°, the predicted dropletsize is 123 μm. In another example, for a channel structure with w=28μm, h=28 μm, and α=7°, the predicted droplet size is 124 μm.

In some instances, the expansion angle, α, may be between a range offrom about 0.5° to about 4°, from about 0.1° to about 10°, or from about0° to about 90°. For example, the expansion angle can be at least about0.01°, 0.1°, 0.2°, 0.3°, 0.4°, 0.5°, 0.6°, 0.7°, 0.8°, 0.9°, 1°, 2°, 3°,4°, 5°, 6°, 7°, 8°, 9°, 10°, 15°, 20°, 25°, 30°, 35°, 40°, 45°, 50°,55°, 60°, 65°, 70°, 75°, 80°, 85°, or higher. In some instances, theexpansion angle can be at most about 89°, 88°, 87°, 86°, 85°, 84°, 83°,82°, 81°, 80°, 75°, 70°, 65°, 60°, 55°, 50°, 45°, 40°, 35°, 30°, 25°,20°, 15°, 10°, 9°, 8°, 7°, 6°, 5°, 4°, 3°, 2°, 1°, 0.1°, 0.01°, or less.In some instances, the width, w, can be between a range of from about100 micrometers (μm) to about 500 μm. In some instances, the width, w,can be between a range of from about 10 μm to about 200 μm.Alternatively, the width can be less than about 10 μm. Alternatively,the width can be greater than about 500 μm. In some instances, the flowrate of the aqueous fluid 1408 entering the junction 1406 can be betweenabout 0.04 microliters (μL)/minute (min) and about 40 μL/min. In someinstances, the flow rate of the aqueous fluid 1408 entering the junction1406 can be between about 0.01 microliters (μL)/minute (min) and about100 μL/min. Alternatively, the flow rate of the aqueous fluid 1408entering the junction 1406 can be less than about 0.01 μL/min.Alternatively, the flow rate of the aqueous fluid 1408 entering thejunction 1406 can be greater than about 40 μL/min, such as 45 μL/min, 50μL/min, 55 μL/min, 60 μL/min, 65 μL/min, 70 μL/min, 75 μL/min, 80μL/min, 85 μL/min, 90 μL/min, 95 μL/min, 100 μL/min, 110 μL, 120 μL/min,130 μL/min, 140 μL/min, 150 μL/min, or greater. At lower flow rates,such as flow rates of about less than or equal to 10 microliters/minute,the droplet radius may not be dependent on the flow rate of the aqueousfluid 1408 entering the junction 1406.

In some instances, at least about 50% of the droplets generated can haveuniform size. In some instances, at least about 55%, 60%, 65%, 70%, 75%,80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or greater of the dropletsgenerated can have uniform size. Alternatively, less than about 50% ofthe droplets generated can have uniform size.

The throughput of droplet generation can be increased by increasing thepoints of generation, such as increasing the number of junctions (e.g.,junction 1406) between aqueous fluid 1408 channel segments (e.g.,channel segment 1402) and the reservoir 1404. Alternatively or inaddition, the throughput of droplet generation can be increased byincreasing the flow rate of the aqueous fluid 1408 in the channelsegment 1402.

FIG. 15 shows an example of a microfluidic channel structure forincreased droplet generation throughput. A microfluidic channelstructure 1500 can comprise a plurality of channel segments 1502 and areservoir 1504. Each of the plurality of channel segments 1502 may be influid communication with the reservoir 1504. The channel structure 1500can comprise a plurality of channel junctions 1506 between the pluralityof channel segments 1502 and the reservoir 1504. Each channel junctioncan be a point of droplet generation. The channel segment 1402 from thechannel structure 1400 in FIG. 14 and any description to the componentsthereof may correspond to a given channel segment of the plurality ofchannel segments 1502 in channel structure 1500 and any description tothe corresponding components thereof. The reservoir 1404 from thechannel structure 1400 and any description to the components thereof maycorrespond to the reservoir 1504 from the channel structure 1500 and anydescription to the corresponding components thereof.

Each channel segment of the plurality of channel segments 1502 maycomprise an aqueous fluid 1508 that includes suspended beads 1512. Thereservoir 1504 may comprise a second fluid 1510 that is immiscible withthe aqueous fluid 1508. In some instances, the second fluid 1510 may notbe subjected to and/or directed to any flow in or out of the reservoir1504. For example, the second fluid 1510 may be substantially stationaryin the reservoir 1504. In some instances, the second fluid 1510 may besubjected to flow within the reservoir 1504, but not in or out of thereservoir 1504, such as via application of pressure to the reservoir1504 and/or as affected by the incoming flow of the aqueous fluid 1508at the junctions. Alternatively, the second fluid 1510 may be subjectedand/or directed to flow in or out of the reservoir 1504. For example,the reservoir 1504 can be a channel directing the second fluid 1510 fromupstream to downstream, transporting the generated droplets.

In operation, the aqueous fluid 1508 that includes suspended beads 1512may be transported along the plurality of channel segments 1502 into theplurality of junctions 1506 to meet the second fluid 1510 in thereservoir 1504 to create droplets 1516, 1518. A droplet may form fromeach channel segment at each corresponding junction with the reservoir1504. At the junction where the aqueous fluid 1508 and the second fluid1510 meet, droplets can form based on factors such as the hydrodynamicforces at the junction, flow rates of the two fluids 1508, 1510, fluidproperties, and certain geometric parameters (e.g., w, h₀, α, etc.) ofthe channel structure 1500, as described elsewhere herein. A pluralityof droplets can be collected in the reservoir 1504 by continuouslyinjecting the aqueous fluid 1508 from the plurality of channel segments1502 through the plurality of junctions 1506. Throughput maysignificantly increase with the parallel channel configuration ofchannel structure 1500. For example, a channel structure having fiveinlet channel segments comprising the aqueous fluid 1508 may generatedroplets five times as frequently than a channel structure having oneinlet channel segment, provided that the fluid flow rate in the channelsegments are substantially the same. The fluid flow rate in thedifferent inlet channel segments may or may not be substantially thesame. A channel structure may have as many parallel channel segments asis practical and allowed for the size of the reservoir. For example, thechannel structure may have at least about 2, 3, 4, 5, 6, 7, 8, 9, 10,20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450,500, 600, 700, 800, 900, 1000, 1500, 5000 or more parallel orsubstantially parallel channel segments.

The geometric parameters, w, h₀, and α, may or may not be uniform foreach of the channel segments in the plurality of channel segments 1502.For example, each channel segment may have the same or different widthsat or near its respective channel junction with the reservoir 1504. Forexample, each channel segment may have the same or different height ator near its respective channel junction with the reservoir 1504. Inanother example, the reservoir 1504 may have the same or differentexpansion angle at the different channel junctions with the plurality ofchannel segments 1502. When the geometric parameters are uniform,beneficially, droplet size may also be controlled to be uniform evenwith the increased throughput. In some instances, when it is desirableto have a different distribution of droplet sizes, the geometricparameters for the plurality of channel segments 1502 may be variedaccordingly.

In some instances, at least about 50% of the droplets generated can haveuniform size. In some instances, at least about 55%, 60%, 65%, 70%, 75%,80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or greater of the dropletsgenerated can have uniform size. Alternatively, less than about 50% ofthe droplets generated can have uniform size.

FIG. 16 shows another example of a microfluidic channel structure forincreased droplet generation throughput. A microfluidic channelstructure 1600 can comprise a plurality of channel segments 1602arranged generally circularly around the perimeter of a reservoir 1604.Each of the plurality of channel segments 1602 may be in fluidcommunication with the reservoir 1604. The channel structure 1600 cancomprise a plurality of channel junctions 1606 between the plurality ofchannel segments 1602 and the reservoir 1604. Each channel junction canbe a point of droplet generation. The channel segment 1402 from thechannel structure 1400 in FIG. 14 and any description to the componentsthereof may correspond to a given channel segment of the plurality ofchannel segments 1602 in channel structure 1600 and any description tothe corresponding components thereof. The reservoir 1404 from thechannel structure 1400 and any description to the components thereof maycorrespond to the reservoir 1604 from the channel structure 1600 and anydescription to the corresponding components thereof.

Each channel segment of the plurality of channel segments 1602 maycomprise an aqueous fluid 1608 that includes suspended beads 1612. Thereservoir 1604 may comprise a second fluid 1610 that is immiscible withthe aqueous fluid 1608. In some instances, the second fluid 1610 may notbe subjected to and/or directed to any flow in or out of the reservoir1604. For example, the second fluid 1610 may be substantially stationaryin the reservoir 1604. In some instances, the second fluid 1610 may besubjected to flow within the reservoir 1604, but not in or out of thereservoir 1604, such as via application of pressure to the reservoir1604 and/or as affected by the incoming flow of the aqueous fluid 1608at the junctions. Alternatively, the second fluid 1610 may be subjectedand/or directed to flow in or out of the reservoir 1604. For example,the reservoir 1604 can be a channel directing the second fluid 1610 fromupstream to downstream, transporting the generated droplets.

In operation, the aqueous fluid 1608 that includes suspended beads 1612may be transported along the plurality of channel segments 1602 into theplurality of junctions 1606 to meet the second fluid 1610 in thereservoir 1604 to create a plurality of droplets 1616. A droplet mayform from each channel segment at each corresponding junction with thereservoir 1604. At the junction where the aqueous fluid 1608 and thesecond fluid 1610 meet, droplets can form based on factors such as thehydrodynamic forces at the junction, flow rates of the two fluids 1608,1610, fluid properties, and certain geometric parameters (e.g., widthsand heights of the channel segments 1602, expansion angle of thereservoir 1604, etc.) of the channel structure 1600, as describedelsewhere herein. A plurality of droplets can be collected in thereservoir 1604 by continuously injecting the aqueous fluid 1608 from theplurality of channel segments 1602 through the plurality of junctions1606. Throughput may significantly increase with the substantiallyparallel channel configuration of the channel structure 1600. A channelstructure may have as many substantially parallel channel segments as ispractical and allowed for by the size of the reservoir. For example, thechannel structure may have at least about 2, 3, 4, 5, 6, 7, 8, 9, 10,20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450,500, 600, 700, 800, 900, 1000, 1500, 5000 or more parallel orsubstantially parallel channel segments. The plurality of channelsegments may be substantially evenly spaced apart, for example, aroundan edge or perimeter of the reservoir. Alternatively, the spacing of theplurality of channel segments may be uneven.

The reservoir 1604 may have an expansion angle, a (not shown in FIG. 16)at or near each channel junction. Each channel segment of the pluralityof channel segments 1602 may have a width, w, and a height, h₀, at ornear the channel junction. The geometric parameters, w, h₀, and α, mayor may not be uniform for each of the channel segments in the pluralityof channel segments 1602. For example, each channel segment may have thesame or different widths at or near its respective channel junction withthe reservoir 1604. For example, each channel segment may have the sameor different height at or near its respective channel junction with thereservoir 1604.

The reservoir 1604 may have the same or different expansion angle at thedifferent channel junctions with the plurality of channel segments 1602.For example, a circular reservoir (as shown in FIG. 16) may have aconical, dome-like, or hemispherical ceiling (e.g., top wall) to providethe same or substantially same expansion angle for each channel segments1602 at or near the plurality of channel junctions 1606. When thegeometric parameters are uniform, beneficially, resulting droplet sizemay be controlled to be uniform even with the increased throughput. Insome instances, when it is desirable to have a different distribution ofdroplet sizes, the geometric parameters for the plurality of channelsegments 1602 may be varied accordingly.

In some instances, at least about 50% of the droplets generated can haveuniform size. In some instances, at least about 55%, 60%, 65%, 70%, 75%,80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or greater of the dropletsgenerated can have uniform size. Alternatively, less than about 50% ofthe droplets generated can have uniform size. The beads and/orbiological particle injected into the droplets may or may not haveuniform size.

FIG. 17A shows a cross-section view of another example of a microfluidicchannel structure with a geometric feature for controlled partitioning.A channel structure 1700 can include a channel segment 1702communicating at a channel junction 1706 (or intersection) with areservoir 1704. In some instances, the channel structure 1700 and one ormore of its components can correspond to any other channel structuredescribed herein and one or more of its components. FIG. 17B shows aperspective view of the channel structure 1700 of FIG. 17A.

An aqueous fluid 1712 comprising a plurality of particles 1716 may betransported along the channel segment 1702 into the junction 1706 tomeet a second fluid 1714 (e.g., oil, etc.) that is immiscible with theaqueous fluid 1712 in the reservoir 1704 to create droplets 720 of theaqueous fluid 1712 flowing into the reservoir 1704. At the junction 1706where the aqueous fluid 1712 and the second fluid 1714 meet, dropletscan form based on factors such as the hydrodynamic forces at thejunction 1706, relative flow rates of the two fluids 1712, 1714, fluidproperties, and certain geometric parameters (e.g., Δh, etc.) of thechannel structure 1700. A plurality of droplets can be collected in thereservoir 1704 by continuously injecting the aqueous fluid 1712 from thechannel segment 1702 at the junction 1706.

A discrete droplet generated may comprise one or more particles of theplurality of particles 1716. As described elsewhere herein, a particlemay be any particle, such as a bead, cell bead, gel bead, biologicalparticle, macromolecular constituents of biological particle, or otherparticles. Alternatively, a discrete droplet generated may not includeany particles.

In some instances, the aqueous fluid 1712 can have a substantiallyuniform concentration or frequency of particles 1716. As describedelsewhere herein (e.g., with reference to FIG. 14), the particles 1716(e.g., beads) can be introduced into the channel segment 1702 from aseparate channel (not shown in FIG. 17). The frequency of particles 1716in the channel segment 1702 may be controlled by controlling thefrequency in which the particles 1716 are introduced into the channelsegment 1702 and/or the relative flow rates of the fluids in the channelsegment 1702 and the separate channel. In some instances, the particles1716 can be introduced into the channel segment 1702 from a plurality ofdifferent channels, and the frequency controlled accordingly. In someinstances, different particles may be introduced via separate channels.For example, a first separate channel can introduce beads and a secondseparate channel can introduce biological particles into the channelsegment 1702. The first separate channel introducing the beads may beupstream or downstream of the second separate channel introducing thebiological particles.

In some instances, the second fluid 1714 may not be subjected to and/ordirected to any flow in or out of the reservoir 1704. For example, thesecond fluid 1714 may be substantially stationary in the reservoir 1704.In some instances, the second fluid 1714 may be subjected to flow withinthe reservoir 1704, but not in or out of the reservoir 1704, such as viaapplication of pressure to the reservoir 1704 and/or as affected by theincoming flow of the aqueous fluid 1712 at the junction 1706.Alternatively, the second fluid 1714 may be subjected and/or directed toflow in or out of the reservoir 1704. For example, the reservoir 1704can be a channel directing the second fluid 1714 from upstream todownstream, transporting the generated droplets.

The channel structure 1700 at or near the junction 1706 may have certaingeometric features that at least partly determine the sizes and/orshapes of the droplets formed by the channel structure 1700. The channelsegment 1702 can have a first cross-section height, h₁, and thereservoir 1704 can have a second cross-section height, h₂. The firstcross-section height, h₁, and the second cross-section height, h₂, maybe different, such that at the junction 1706, there is a heightdifference of Δh. The second cross-section height, h₂, may be greaterthan the first cross-section height, h₁. In some instances, thereservoir may thereafter gradually increase in cross-section height, forexample, the more distant it is from the junction 1706. In someinstances, the cross-section height of the reservoir may increase inaccordance with expansion angle, β, at or near the junction 1706. Theheight difference, Δh, and/or expansion angle, β, can allow the tongue(portion of the aqueous fluid 1712 leaving channel segment 1702 atjunction 1706 and entering the reservoir 1704 before droplet formation)to increase in depth and facilitate decrease in curvature of theintermediately formed droplet. For example, droplet size may decreasewith increasing height difference and/or increasing expansion angle.

The height difference, Δh, can be at least about 1 μm. Alternatively,the height difference can be at least about 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 60,70, 80, 90, 100, 200, 300, 400, 500 μm or more. Alternatively, theheight difference can be at most about 500, 400, 300, 200, 100, 90, 80,70, 60, 50, 45, 40, 35, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11,10, 9, 8, 7, 6, 5, 4, 3, 2, 1 μm or less. In some instances, theexpansion angle, β, may be between a range of from about 0.5° to about4°, from about 0.1° to about 10°, or from about 0° to about 90°. Forexample, the expansion angle can be at least about 0.01°, 0.1°, 0.2°,0.3°, 0.4°, 0.5°, 0.6°, 0.7°, 0.8°, 0.9°, 1°, 2°, 3°, 4°, 5°, 6°, 7°,8°, 9°, 10°, 15°, 20°, 25°, 30°, 35°, 40°, 45°, 50°, 55°, 60°, 65°, 70°,75°, 80°, 85°, or higher. In some instances, the expansion angle can beat most about 89°, 88°, 87°, 86°, 85°, 84°, 83°, 82°, 81°, 80°, 75°,70°, 65°, 60°, 55°, 50°, 45°, 40°, 35°, 30°, 25°, 20°, 15°, 10°, 9°, 8°,7°, 6°, 5°, 4°, 3°, 2°, 1°, 0.1°, 0.01°, or less.

In some instances, the flow rate of the aqueous fluid 1712 entering thejunction 1706 can be between about 0.04 microliters (μL)/minute (min)and about 40 μL/min. In some instances, the flow rate of the aqueousfluid 1712 entering the junction 1706 can be between about 0.01microliters (μL)/minute (min) and about 100 μL/min. Alternatively, theflow rate of the aqueous fluid 1712 entering the junction 1706 can beless than about 0.01 μL/min. Alternatively, the flow rate of the aqueousfluid 1712 entering the junction 1706 can be greater than about 40μL/min, such as 45 μL/min, 50 μL/min, 55 μL/min, 60 μL/min, 65 μL/min,70 μL/min, 75 μL/min, 80 μL/min, 85 μL/min, 90 μL/min, 95 μL/min, 100μL/min, 110 μL/min, 120 μL/min, 130 μL/min, 140 μL/min, 150 μL/min, orgreater. At lower flow rates, such as flow rates of about less than orequal to 10 microliters/minute, the droplet radius may not be dependenton the flow rate of the aqueous fluid 1712 entering the junction 1706.The second fluid 1714 may be stationary, or substantially stationary, inthe reservoir 1704. Alternatively, the second fluid 1714 may be flowing,such as at the above flow rates described for the aqueous fluid 1712.

In some instances, at least about 50% of the droplets generated can haveuniform size. In some instances, at least about 55%, 60%, 65%, 70%, 75%,80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or greater of the dropletsgenerated can have uniform size. Alternatively, less than about 50% ofthe droplets generated can have uniform size.

While FIGS. 7A and 7B illustrate the height difference, Δh, being abruptat the junction 1706 (e.g., a step increase), the height difference mayincrease gradually (e.g., from about 0 μm to a maximum heightdifference). Alternatively, the height difference may decrease gradually(e.g., taper) from a maximum height difference. A gradual increase ordecrease in height difference, as used herein, may refer to a continuousincremental increase or decrease in height difference, wherein an anglebetween any one differential segment of a height profile and animmediately adjacent differential segment of the height profile isgreater than 90°. For example, at the junction 1706, a bottom wall ofthe channel and a bottom wall of the reservoir can meet at an anglegreater than 90°. Alternatively or in addition, a top wall (e.g.,ceiling) of the channel and a top wall (e.g., ceiling) of the reservoircan meet an angle greater than 90°. A gradual increase or decrease maybe linear or non-linear (e.g., exponential, sinusoidal, etc.).Alternatively or in addition, the height difference may variablyincrease and/or decrease linearly or non-linearly. While FIGS. 7A and 7Billustrate the expanding reservoir cross-section height as linear (e.g.,constant expansion angle, β), the cross-section height may expandnon-linearly. For example, the reservoir may be defined at leastpartially by a dome-like (e.g., hemispherical) shape having variableexpansion angles. The cross-section height may expand in any shape.

The channel networks, e.g., as described above or elsewhere herein, canbe fluidly coupled to appropriate fluidic components. For example, theinlet channel segments are fluidly coupled to appropriate sources of thematerials they are to deliver to a channel junction. These sources mayinclude any of a variety of different fluidic components, from simplereservoirs defined in or connected to a body structure of a microfluidicdevice, to fluid conduits that deliver fluids from off-device sources,manifolds, fluid flow units (e.g., actuators, pumps, compressors) or thelike. Likewise, the outlet channel segment (e.g., channel segment 1208,reservoir 1604, etc.) may be fluidly coupled to a receiving vessel orconduit for the partitioned cells for subsequent processing. Again, thismay be a reservoir defined in the body of a microfluidic device, or itmay be a fluidic conduit for delivering the partitioned cells to asubsequent process operation, instrument or component.

The methods and systems described herein may be used to greatly increasethe efficiency of single cell applications and/or other applicationsreceiving droplet-based input. For example, following the sorting ofoccupied cells and/or appropriately-sized cells, subsequent operationsthat can be performed can include generation of amplification products,purification (e.g., via solid phase reversible immobilization (SPRI)),further processing (e.g., shearing, ligation of functional sequences,and subsequent amplification (e.g., via PCR)). These operations mayoccur in bulk (e.g., outside the partition). In the case where apartition is a droplet in an emulsion, the emulsion can be broken andthe contents of the droplet pooled for additional operations. Additionalreagents that may be co-partitioned along with the barcode bearing beadmay include oligonucleotides to block ribosomal RNA (rRNA) and nucleasesto digest genomic DNA from cells. Alternatively, rRNA removal agents maybe applied during additional processing operations. The configuration ofthe constructs generated by such a method can help minimize (or avoid)sequencing of the poly-T sequence during sequencing and/or sequence the5′ end of a polynucleotide sequence. The amplification products, forexample, first amplification products and/or second amplificationproducts, may be subject to sequencing for sequence analysis. In somecases, amplification may be performed using the Partial HairpinAmplification for Sequencing (PHASE) method.

A variety of applications require the evaluation of the presence andquantification of different biological particle or organism types withina population of biological particles, including, for example, microbiomeanalysis and characterization, environmental testing, food safetytesting, epidemiological analysis, e.g., in tracing contamination or thelike.

Computer Control Systems

The present disclosure provides computer control systems that areprogrammed to implement methods of the disclosure. FIG. 18 shows acomputer system 1801 that is programmed or otherwise configured to (i)sort occupied droplets from unoccupied droplets by includingfield-attractable particles in each droplet and applying a force field,(ii) sort occupied droplets from unoccupied droplets by applying apressure pulse, (iii) sort occupied particles (e.g., cell beads) fromunoccupied particles using field-attractable particles by applying aforce field, (iv) sort occupied particles (e.g., cell beads) fromunoccupied particles by applying a pressure pulse, (v) selectivelypolymerize occupied droplets, and/or (vi) selectively polymerizeappropriately sized droplets. The computer system 1801 can regulatevarious aspects of the present disclosure, such as, for example, thetimed exposure of the single biological particle to a variety ofchemical or biological operations, regulating fluid flow rate in one ormore channels in a microfluidic structure, regulating field strengthapplied by one or more field application units, regulating pressurepulses applied by one or more pressure application units, and/orregulating timing of polymerization application units. The computersystem 1801 can be an electronic device of a user or a computer systemthat is remotely located with respect to the electronic device. Theelectronic device can be a mobile electronic device.

The computer system 1801 includes a central processing unit (CPU, also“processor” and “computer processor” herein) 1805, which can be a singlecore or multi core processor, or a plurality of processors for parallelprocessing. The computer system 1801 also includes memory or memorylocation 1810 (e.g., random-access memory, read-only memory, flashmemory), electronic storage unit 1815 (e.g., hard disk), communicationinterface 1820 (e.g., network adapter) for communicating with one ormore other systems, and peripheral devices 1825, such as cache, othermemory, data storage and/or electronic display adapters. The memory1810, storage unit 1815, interface 1820 and peripheral devices 1825 arein communication with the CPU 1805 through a communication bus (solidlines), such as a motherboard. The storage unit 1815 can be a datastorage unit (or data repository) for storing data. The computer system1801 can be operatively coupled to a computer network (“network”) 1830with the aid of the communication interface 1820. The network 1830 canbe the Internet, an internet and/or extranet, or an intranet and/orextranet that is in communication with the Internet. The network 1830 insome cases is a telecommunication and/or data network. The network 1830can include one or more computer servers, which can enable distributedcomputing, such as cloud computing. The network 1830, in some cases withthe aid of the computer system 1801, can implement a peer-to-peernetwork, which may enable devices coupled to the computer system 1801 tobehave as a client or a server.

The CPU 1805 can execute a sequence of machine-readable instructions,which can be embodied in a program or software. The instructions may bestored in a memory location, such as the memory 1810. The instructionscan be directed to the CPU 1805, which can subsequently program orotherwise configure the CPU 1805 to implement methods of the presentdisclosure. Examples of operations performed by the CPU 1805 can includefetch, decode, execute, and writeback.

The CPU 1805 can be part of a circuit, such as an integrated circuit.One or more other components of the system 1801 can be included in thecircuit. In some cases, the circuit is an application specificintegrated circuit (ASIC).

The storage unit 1815 can store files, such as drivers, libraries andsaved programs. The storage unit 1815 can store user data, e.g., userpreferences and user programs. The computer system 1801 in some casescan include one or more additional data storage units that are externalto the computer system 1801, such as located on a remote server that isin communication with the computer system 1801 through an intranet orthe Internet.

The computer system 1801 can communicate with one or more remotecomputer systems through the network 1830. For instance, the computersystem 1801 can communicate with a remote computer system of a user(e.g., operator). Examples of remote computer systems include personalcomputers (e.g., portable PC), slate or tablet PC's (e.g., Apple® iPad,Samsung® Galaxy Tab), telephones, Smart phones (e.g., Apple® iPhone,Android-enabled device, Blackberry®), or personal digital assistants.The user can access the computer system 1801 via the network 1830.

Methods as described herein can be implemented by way of machine (e.g.,computer processor) executable code stored on an electronic storagelocation of the computer system 1801, such as, for example, on thememory 1810 or electronic storage unit 1815. The machine executable ormachine readable code can be provided in the form of software. Duringuse, the code can be executed by the processor 1805. In some cases, thecode can be retrieved from the storage unit 1815 and stored on thememory 1810 for ready access by the processor 1805. In some situations,the electronic storage unit 1815 can be precluded, andmachine-executable instructions are stored on memory 1810.

The code can be pre-compiled and configured for use with a machinehaving a processor adapted to execute the code, or can be compiledduring runtime. The code can be supplied in a programming language thatcan be selected to enable the code to execute in a pre-compiled oras-compiled fashion.

Aspects of the systems and methods provided herein, such as the computersystem 1801, can be embodied in programming. Various aspects of thetechnology may be thought of as “products” or “articles of manufacture”typically in the form of machine (or processor) executable code and/orassociated data that is carried on or embodied in a type of machinereadable medium. Machine-executable code can be stored on an electronicstorage unit, such as memory (e.g., read-only memory, random-accessmemory, flash memory) or a hard disk. “Storage” type media can includeany or all of the tangible memory of the computers, processors or thelike, or associated modules thereof, such as various semiconductormemories, tape drives, disk drives and the like, which may providenon-transitory storage at any time for the software programming. All orportions of the software may at times be communicated through theInternet or various other telecommunication networks. Suchcommunications, for example, may enable loading of the software from onecomputer or processor into another, for example, from a managementserver or host computer into the computer platform of an applicationserver. Thus, another type of media that may bear the software elementsincludes optical, electrical and electromagnetic waves, such as usedacross physical interfaces between local devices, through wired andoptical landline networks and over various air-links. The physicalelements that carry such waves, such as wired or wireless links, opticallinks or the like, also may be considered as media bearing the software.As used herein, unless restricted to non-transitory, tangible “storage”media, terms such as computer or machine “readable medium” refer to anymedium that participates in providing instructions to a processor forexecution.

Hence, a machine readable medium, such as computer-executable code, maytake many forms, including but not limited to, a tangible storagemedium, a carrier wave medium or physical transmission medium.Non-volatile storage media include, for example, optical or magneticdisks, such as any of the storage devices in any computer(s) or thelike, such as may be used to implement the databases, etc. shown in thedrawings. Volatile storage media include dynamic memory, such as mainmemory of such a computer platform. Tangible transmission media includecoaxial cables; copper wire and fiber optics, including the wires thatcomprise a bus within a computer system. Carrier-wave transmission mediamay take the form of electric or electromagnetic signals, or acoustic orlight waves such as those generated during radio frequency (RF) andinfrared (IR) data communications. Common forms of computer-readablemedia therefore include for example: a floppy disk, a flexible disk,hard disk, magnetic tape, any other magnetic medium, a CD-ROM, DVD orDVD-ROM, any other optical medium, punch cards paper tape, any otherphysical storage medium with patterns of holes, a RAM, a ROM, a PROM andEPROM, a FLASH-EPROM, any other memory chip or cartridge, a carrier wavetransporting data or instructions, cables or links transporting such acarrier wave, or any other medium from which a computer may readprogramming code and/or data. Many of these forms of computer readablemedia may be involved in carrying one or more sequences of one or moreinstructions to a processor for execution.

The computer system 1801 can include or be in communication with anelectronic display 1835 that comprises a user interface (UI) 1840 forproviding, for example, fluid control options (e.g., fluid flow rate,timing of applying polymerization source (e.g., light), strength ofmagnetic of electric force field, strength and/or frequency of pressurepulses, etc.). Examples of UI's include, without limitation, a graphicaluser interface (GUI) and web-based user interface.

Methods and systems of the present disclosure can be implemented by wayof one or more algorithms. An algorithm can be implemented by way ofsoftware upon execution by the central processing unit 1805. Thealgorithm can, for example, (i) sort occupied droplets from unoccupieddroplets by including field-attractable particles in each droplet andapplying a force field, (ii) sort occupied droplets from unoccupieddroplets by applying a pressure pulse, (iii) sort occupied particles(e.g., cell beads) from unoccupied particles using field-attractableparticles by applying a force field, (iv) sort occupied particles (e.g.,cell beads) from unoccupied particles by applying a pressure pulse, (v)selectively polymerize occupied droplets, and/or (vi) selectivelypolymerize appropriately sized droplets. The algorithm can also, forexample, generate a plurality of droplets that may or may not containbiological particles (cells) and/or barcode carrying beads (particles).

While preferred embodiments of the present invention have been shown anddescribed herein, it will be obvious to those skilled in the art thatsuch embodiments are provided by way of example only. It is not intendedthat the invention be limited by the specific examples provided withinthe specification. While the invention has been described with referenceto the aforementioned specification, the descriptions and illustrationsof the embodiments herein are not meant to be construed in a limitingsense. Numerous variations, changes, and substitutions will now occur tothose skilled in the art without departing from the invention.Furthermore, it shall be understood that all aspects of the inventionare not limited to the specific depictions, configurations or relativeproportions set forth herein which depend upon a variety of conditionsand variables. It should be understood that various alternatives to theembodiments of the invention described herein may be employed inpracticing the invention. It is therefore contemplated that theinvention shall also cover any such alternatives, modifications,variations or equivalents. It is intended that the following claimsdefine the scope of the invention and that methods and structures withinthe scope of these claims and their equivalents be covered thereby.

What is claimed is:
 1. A method for sorting droplets, comprising: (a)bringing a first phase in contact with a second phase immiscible withsaid first phase, to generate a plurality of droplets, wherein saidplurality of droplets comprises (i) a first subset of droplets eachincluding, and not more than, one biological particle, and (ii) a secondsubset of droplets each either having more than one biological particleor not having any biological particle, wherein said biological particleis a cell, a derivative of a cell, or a constituent of a cell; whereineach of said plurality of droplets comprises field-attractableparticles; and wherein a concentration of said field-attractableparticles in droplets of said second subset not having any biologicalparticle is substantially uniform; (b) directing said plurality ofdroplets along a first channel towards an intersection of said firstchannel with at least a second channel and a third channel; and (c)subjecting said plurality of droplets to an electric or magnetic fieldunder conditions sufficient to separate said at least said portion ofsaid first subset from said at least said portion of said second subset,thereby separating at least a portion of said first subset of saidplurality of droplets from at least a portion of said second subset ofsaid plurality of droplets, wherein upon separation, said at least saidportion of said first subset of said plurality of droplets flows alongsaid second channel and said at least said portion of said second subsetof said plurality of droplets flows along said third channel.
 2. Themethod of claim 1, wherein said first subset of said plurality ofdroplets includes particles having coupled thereto molecules comprisingbarcode sequences.
 3. The method of claim 2, wherein said particles aregel beads.
 4. The method of claim 1, further comprising detectingindividual droplets of said first subset of said plurality of droplets,and subjecting said individual droplets to a stimulus to facilitatepolymerization in said biological particles upon detecting saidindividual droplets.
 5. The method of claim 4 wherein said stimulus isapplied prior to said intersection.
 6. The method of claim 4, whereinsaid stimulus is applied subsequent to said intersection.
 7. The methodof claim 4, wherein said stimulus is an optical stimulus or chemicalstimulus.
 8. The method of claim 1, wherein said biological particlesare cells enclosed within or comprising a gel or polymer matrix.
 9. Amethod for sorting droplets, comprising: (a) bringing a first phase incontact with a second phase immiscible with said first phase, togenerate a plurality of droplets, wherein said plurality of dropletscomprises (i) a first subset of droplets each including, and not morethan, one biological particle, and (ii) a second subset of droplets eacheither having more than one biological particle or not having anybiological particle, wherein said biological particle is a cell, aderivative of a cell, or a constituent of a cell; wherein each of saidplurality of droplets comprises field-attractable particles; and whereineach droplet of said first subset comprises (i) less field attractableparticles than each droplet of said second subset not having anybiological particle, and (ii) more field attractable particles than eachdroplet of said second subset having more than one biological particle;(b) directing said plurality of droplets along a first channel towardsan intersection of said first channel with at least a second channel anda third channel; and (c) subjecting said plurality of droplets to anelectric or magnetic field under conditions sufficient to separate saidat least said portion of said first subset from said at least saidportion of said second subset, thereby separating at least a portion ofsaid first subset of said plurality of droplets from at least a portionof said second subset of said plurality of droplets, wherein uponseparation, said at least said portion of said first subset of saidplurality of droplets flows along said second channel and said at leastsaid portion of said second subset of said plurality of droplets flowsalong said third channel.
 10. The method of claim 9, wherein forcesinduced by said electric or magnetic field on droplets of said secondsubset not having any biological particle are greater than forcesinduced on said first subset, and wherein forces induced on said firstsubset are greater than forces induced on droplets of said second subsethaving more than one biological particle.
 11. The method of claim 1,wherein said field-attractable particles are magnetic-field attractableparticles.
 12. A method for sorting droplets, comprising: (a) bringing afirst phase in contact with a second phase immiscible with said firstphase, to generate a plurality of droplets, wherein said plurality ofdroplets comprises (i) a first subset of droplets each including, andnot more than, one biological particle, and (ii) a second subset ofdroplets each either having more than one biological particle or nothaving any biological particle, wherein said biological particle is acell, a derivative of a cell, or a constituent of a cell; and whereineach of said plurality of droplets comprises field-attractableparticles, (b) directing said plurality of droplets along a firstchannel towards an intersection of said first channel with at least asecond channel and a third channel; and (c) subjecting said plurality ofdroplets to an electric or magnetic field under conditions sufficient toseparate said at least said portion of said first subset from said atleast said portion of said second subset, thereby separating at least aportion of said first subset of said plurality of droplets from at leasta portion of said second subset of said plurality of droplets, whereinupon separation, said at least said portion of said first subset of saidplurality of droplets flows along said second channel and said at leastsaid portion of said second subset of said plurality of droplets flowsalong said third channel; wherein said conditions of said electric ormagnetic field sufficient to separate said at least said portion of saidfirst subset and said at least said portion of said second subset aredetermined based at least in part on a ratio between sizes of saidplurality of droplets and sizes of said biological particles in saidfirst subset of said plurality of droplets.
 13. The method of claim 1,further comprising, subsequent to (c), subjecting nucleic acid moleculesderived from said biological particles in said first subset to nucleicacid sequencing.
 14. The method of claim 13, further comprising,subsequent to (c), subjecting said first subset of said plurality ofdroplets to conditions sufficient to yield extension products of saidnucleic acid molecules from said biological particles in said firstsubset, and subjecting said extension products or derivatives thereof tonucleic acid sequencing.
 15. The method of claim 1, wherein uponseparation, said at least said portion of said first subset and dropletsof said second subset having more than one biological particle flowalong said second channel and droplets of said second subset not havingany biological particle flow along said third channel.
 16. A method forsorting droplets, comprising: (a) bringing a first phase in contact witha second phase immiscible with said first phase, to generate a pluralityof droplets, wherein said plurality of droplets comprises (i) a firstsubset of droplets each including, and not more than, one biologicalparticle, and (ii) a second subset of droplets each either having morethan one biological particle or not having any biological particle,wherein said biological particle is a cell, a derivative of a cell, or aconstituent of a cell; (b) directing said plurality of droplets along afirst channel towards an intersection of said first channel with atleast a second channel and a third channel; and (c) separating at leasta portion of said first subset of said plurality of droplets from atleast a portion of said second subset of said plurality of droplets,wherein upon separation, said at least said portion of said first subsetand said second subset having more than one biological particle flowalong said second channel and droplets of said second subset not havingany biological particle flow along said third channel; (d) directingsaid at least said portion of said first subset and said droplets ofsaid second subset having more than one biological particle along saidsecond channel towards a second intersection of said second channel withat least a fourth channel and a fifth channel; and (e) separating saidat least said portion of said first subset from at least a portion ofsaid droplets of said second subset having more than one biologicalparticle, wherein upon separation, said at least said portion of saidfirst subset flows along said fourth channel and said at least saidportion of said droplets of said second subset having more than onebiological particle flows along said fifth channel.
 17. The method ofclaim 9, wherein said first subset of said plurality of dropletsincludes particles having coupled thereto molecules comprising barcodesequences.
 18. The method of claim 17, wherein said particles are gelbeads.
 19. The method of claim 9, wherein said biological particles arecells enclosed within or comprising a gel or polymer matrix.
 20. Themethod of claim 9, further comprising, subsequent to (c), subjectingnucleic acid molecules derived from said biological particles in saidfirst subset to nucleic acid sequencing.
 21. The method of claim 12,wherein said first subset of said plurality of droplets includesparticles having coupled thereto molecules comprising barcode sequences.22. The method of claim 21, wherein said particles are gel beads. 23.The method of claim 12, wherein said biological particles are cellsenclosed within or comprising a gel or polymer matrix.
 24. The method ofclaim 12, further comprising, subsequent to (c), subjecting nucleic acidmolecules derived from said biological particles in said first subset tonucleic acid sequencing.
 25. The method of claim 16, wherein said firstsubset of said plurality of droplets includes particles having coupledthereto molecules comprising barcode sequences.
 26. The method of claim25, wherein said particles are gel beads.
 27. The method of claim 16,wherein said biological particles are cells enclosed within orcomprising a gel or polymer matrix.
 28. The method of claim 16, furthercomprising, subsequent to (c), subjecting nucleic acid molecules derivedfrom said biological particles in said first subset to nucleic acidsequencing.
 29. The method of claim 16, wherein (c) comprises subjectingsaid plurality of droplets to a pressure pulse under conditionssufficient to separate said at least said portion of said first subsetfrom said at least said portion of said second subset.
 30. The method ofclaim 29, wherein forces induced by said pressure pulse on droplets ofsaid second subset not having any biological particle are greater thanforces induced on said first subset, and wherein forces induced on saidfirst subset are greater than forces induced on droplets of said secondsubset having more than one biological particle.